BACKGROUND
chromosomal dna within eukaryotic cells is constantly subjected to many types of damage from both endogenous and exogenous sources.
repair of the damage may be performed by one or more different pathways, depending on the specific type of dna lesion involved.
major pathways that have been characterized include nucleotide excision repair , active on ultraviolet light induced dna crosslinks and other bulky lesions, base excision repair  for repair of damaged and lost bases, and mismatch repair .
two other pathways, homologous recombination  and nonhomologous end-joining , are active in repair of dna double-strand breaks .
dsbs can be induced by endogenous processes such as oxidation, arrest of dna replication forks, nuclease cleavage or processing of various dna lesions and by exposure to external agents such as ionizing radiation or clastogenic chemicals  <cit> .
dsb ends may be blunt or may contain single-strand overhangs.
the ends may also have other associated damage such as altered or missing bases, e.g., as seen after cells are exposed to ionizing radiation or the chemical bleomycin  <cit> .
most dna damaging agents that induce dsbs generate other lesions as well, with the other lesions typically occurring at much higher frequencies.
despite their rarity, unrepaired and/or misrepaired dsbs are believed to be the major cause of cell death after exposure to such agents  <cit> .
repair of dsbs by nhej and hr involves the concerted actions of many conserved proteins found in both single-celled and multicellular organisms.
in saccharomyces cerevisiae, or budding yeast, nhej requires at least three factors.
the yku70/yku <dig> complex binds to dna ends, provides protection from nucleases, and recruits other repair proteins.
the mre11/rad50/xrs <dig>  complex forms a bridge between the broken dna ends and also has nuclease activities involved in processing of some end structures.
dnl4/lif1/nej <dig> represents the dna ligase iv complex, which covalently links broken dna strands together  <cit> .
nej <dig> may also function earlier in the pathway, modulating the binding of yku70/yku <dig> to dna ends  <cit> .
the chromatin silencing proteins sir <dig>  sir <dig> and sir <dig> are also required in yeast cells, but they primarily affect nhej indirectly through their effects on nej <dig> gene expression  <cit> .
other proteins such as exo <dig>  rad <dig>  pol <dig>  the rsc and smc complexes, and dna damage checkpoint proteins have also been suggested to modulate the efficiency of nhej repair  <cit> .
repair by the homologous recombination pathway involves association of broken dsb ends with an unbroken homologous dna molecule such as a sister chromatid or a homologous chromosome, which then serves as a template for repair.
efficient repair by this pathway requires the functions of many proteins, especially several members of the rad <dig> group   <cit> .
this group of genes was originally defined in the 1970s on the basis of mutant phenotypes.
with the exceptions of rad <dig> and rdh <dig>  which are weak homologues of rad <dig> and rad <dig>  respectively, mutants with defects in these genes share many common phenotypes.
they exhibit strong sensitivity to ionizing radiation and chemical clastogens, weak sensitivity to ultraviolet light, and genetic epistasis when combined with mutations in other members of the group to generate double mutants  <cit> .
two other original members of this group are rad <dig>  now known to function primarily in the dna damage-induced checkpoint response, and rad <dig>  a locus that remains uncharacterized  <cit> .
an additional gene, rad <dig>  has also been suggested as a member, though its main function may lie in sister chromatid cohesion  <cit> .
the general model for recombinational repair of dsbs involves several steps.
first, broken ends are resected by mrx and other enzymes such as exo <dig>  sgs <dig>  sae <dig> and dna <dig> to create long 3’-ended single-stranded dna  tails.
the tails are subsequently bound by the ssdna binding protein complex , rad <dig>  and other proteins that recruit factors promoting a homology search, strand invasion of an unbroken dna molecule, new dna synthesis and other steps ultimately leading to repair of the broken strands and separation  of the paired complexes  <cit> .
several additional proteins have been shown to influence the efficiency of the pathway.
these include components of the dna damage checkpoint response , nucleosome remodeling , sister chromatid cohesion , other end-processing nucleases , resolvase-like enzymes  and chromatin reassembly proteins  <cit> .
the development of yeast deletion strain libraries has allowed researchers to screen thousands of individual gene mutants in a single experiment to identify all mutants that have a particular phenotype.
several previous studies have involved testing such strains for sensitivities to physical or chemical agents that damage dna, including ionizing radiation, ultraviolet light, methyl methanesulfonate  and other chemicals  <cit> .
genomic screens by bennett et al.
and game et al.
using over  <dig> diploid mutants identified a large number of genes that are required for resistance to gamma radiation  <cit> .
ionizing radiation generates low levels of dsbs in cellular dna, but it induces higher frequencies of many other types of dna damage and also causes damage to other macromolecules inside the cell.
thus, only a subset of the genes found to be required for radiation resistance are actually likely to affect dsb repair.
the bacterial restriction endonuclease ecori recognizes and cuts the palindromic 6 bp sequence g^aattc, generating 5’ ssdna overhangs that are  <dig> nt long.
controlled in vivo expression of this nuclease from a galactose-regulated promoter has been employed in several studies investigating cellular responses to dsbs and their repair  <cit> .
use of ecori was advantageous for these studies because it is believed to produce essentially only one type of dna lesion, a dsb.
thus, it is more specific than commonly used clastogens like radiation, mms or bleomycin.
ecori-induced dsbs are repaired efficiently in most wildtype haploid strains of yeast, but produce strong growth inhibition and modest killing in most recombination-deficient rad <dig> group mutants  <cit> .
in contrast, the impact of ecori expression on nhej mutants  is more variable and dependent on the strain background employed for the study.
this characteristic is similar to the variable sensitivities to mms and bleomycin that have been observed in nhej mutants  <cit> .
progress in understanding dsb repair pathways has been hindered because many of the proteins acting on or influencing efficiency at each of the various steps have remained unidentified.
in the current study we tested each of the genes shown previously to be important for resistance to ionizing radiation, using both matα and mata haploid mutant libraries, to identify those genes that specifically affect dsb repair.
this goal was accomplished by screening the mutants for sensitivity to dsbs produced by in vivo expression of ecori.
use of two libraries was advantageous because it allowed phenotypes observed in one haploid mutant to be confirmed in an equivalent mutant of opposite mating type.
these efforts have resulted in the identification and characterization of  <dig> non-rad <dig> group genes that are required for efficient repair of site-specific dsbs.
RESULTS
two previous screens undertaken using diploid yeast deletion strain libraries , identified a total of  <dig> mutants with reduced resistance to gamma radiation  <cit> .
several of these mutants corresponded to known rad <dig> group genes defective in recombination, but most genes had not previously been associated with dna repair.
because radiation generates many types of cellular damage, only a subset of these genes are likely to affect dsb repair.
to identify genes specifically impacting repair of dsbs, haploid matα versions of each of the  <dig> mutants were tested for resistance to dsbs produced by in vivo expression of ecori endonuclease from a gal <dig> promoter.
one more library mutant, exo <dig>  was added to the screen, bringing the total to  <dig>  exo <dig> is a 5’-to’3’ exonuclease active in resection of dsb ends in the homologous recombination pathway and it has other roles in replication and mismatch repair  <cit> .
although exo1−/− cells were not detected in the original diploid screens, we have previously observed that exo <dig> haploid library mutants are gamma-sensitive  and human exo1−/− cells are sensitive to ionizing radiation  <cit> .
for these experiments, growth of cells containing the control vector prs <dig> or the gal1p::ecori fusion plasmid ycpgal::rib  <cit>  was initially analyzed by “double-imprint” replica-plating patches of cells from plates containing 2% raffinose to plates containing 2% galactose .
mutants whose growth was inhibited in the presence of galactose were then retested using more quantitative dilution pronging survival assays.
for each pronging assay, cells containing either the vector or the gal1p::ecori plasmid were harvested from patches on raffinose plates, serially diluted 5-fold into 96-well microtiter dishes, and pronged to plates containing raffinose or galactose.
tests of control matα haploid library strains revealed that survival of wildtype by <dig> cells expressing ecori was high.
in contrast, cell survival  and growth rates  were reduced in several rad <dig> group recombination mutants  .
colony formation in mutants defective in nhej repair  was similar to that of wildtype cells in this strain  .
this phenotype was not unexpected because dna damage sensitivities of nhej mutants are known to vary in different strain backgrounds  <cit> .
testing of the  <dig> haploid matα library mutants revealed that survival of many of the strains was reduced by expression of ecori.
results obtained with two mutants, mms <dig> and mms <dig>  are depicted in figure 1b along with wt and mre <dig> strain controls.
interestingly,  <dig> of the  <dig> mutants were successfully evaluated using the procedure described above, but  <dig> of the matα library strains could not be tested, usually because they grew too poorly on galactose plates.
some mutants could not be tested for other reasons.
ade <dig> mutants did not produce ura+ colonies even after repeated transformations with prs <dig> and ycpgal:rib plasmid dnas.
also, cdc <dig> cells from the library were phenotypically ura+, preventing use of the ura <dig> selectable marker on the two plasmids.
a total of  <dig> of the  <dig> matα mutants that could be tested exhibited killing when ecori was expressed.
to address the  <dig> matα library strains that were untestable,  <dig> haploid mutants with the equivalent genes inactivated were obtained from an alternative deletion strain collection of opposite mating type.
twenty-two of these  <dig> mata strains grew on galactose media and could be successfully transformed with the assay plasmids.
this result suggests that many of the  <dig> original matα library versions of the strains had secondary mutations affecting carbohydrate metabolism.
only two of the mata mutants, ade <dig> and hfi <dig>  remained untestable.
similar to the matα library strains, transformations of mata ade <dig> mutants did not produce ura+ colonies, and hfi <dig> cells from both libraries grew too poorly to be tested.
eleven of the  <dig> mata mutants were found to be ecori-sensitive .
thus,  <dig> of the original  <dig> genes were tested,  <dig> as matα strains and  <dig> as mata cells, and  <dig> of them were identified as important for normal resistance to ecori-induced dsbs in haploid cells.
as shown in the top section of table  <dig>  eight members of the rad <dig> group  were required for normal resistance to ecori.
deletions of two other group members, rad <dig> and rdh <dig>  had no detectable effect.
another gene proposed to be a member of the group called rad <dig> <cit>  was also not required for repair of the ecori-induced dsbs.
both mata and matα versions of rad <dig>  rdh <dig> and rad <dig> strains were tested and found to be resistant to ecori .
all mutants in table  <dig> were classified as moderately sensitive  or strongly sensitive , based on the extent of colony formation seen in the semi-quantitative pronging survival assays .
a eight rad <dig> group mutants are shown at the top of the table.
mutants were ranked as resistant , sensitive , or strongly sensitive  .
n/d usually indicates that the library strain was unable to grow on galactose plates.
some genes could not be tested for other reasons, e.g., matα cdc <dig> cells were ura+.
b gene names separated by a forward slash indicate deletions affecting two or more overlapping open reading frames.
the coding region of the gene listed first was deleted in each strain.
c the matα mre <dig> strain was reconstructed for this study because the library mutant did not exhibit phenotypes characteristic of mre <dig> mutants .
drtt <dig> is usually referred to as rem <dig> in the literature.
eyml009w-b overlaps the verified gene spt <dig> and another open reading frame called yml009c-a.
because of the apparent high frequency of secondary mutations in the original library, experiments were performed to determine if ecori sensitivities seen in the matα mutants could be confirmed in mata versions of the strains.
the ecoris phenotype was reproduced in  <dig> of the  <dig> equivalent mata strains using dilution pronging as before, with only psy <dig>  rad <dig> and spt <dig> mata library strains exhibiting resistance .
these experiments therefore identified  <dig> rad <dig> group mutants and  <dig> non-rad <dig> group mutants as important for survival after induction of ecori.
for  <dig> of the latter mutants, sensitivity was confirmed in both matα and mata strains.
for completeness, all of the library mutants that were tested in this study and found to be resistant to ecori are listed in additional file 1: table s <dig>
the  <dig> mutants exhibiting sensitivity to ecori were further characterized by assessing their survival after exposure to the chemicals mms and bleomycin.
these chemicals induce dsbs by very different mechanisms and have been widely used to investigate dna repair pathways  <cit> .
the eight rad <dig> group mutants plus all  <dig> other mutants were analyzed using dilution pronging survival assays.
strains from the original matα library were used for the experiments, with mata library strains substituted only for matα cells that had growth defects.
rad <dig> group mutants exhibited strong killing on plates containing either 2 mm mms or 4 μg/ml bleomycin .
rad <dig> and rad <dig> mutants were least sensitive in the assays, which were conducted at 30°c; these mutants exhibit greatest sensitivity to dna damaging agents at 23°c  <cit> .
higher concentrations of mms and bleomycin were not used because they caused strong growth inhibition of wt cells .
many of the  <dig> non-rad <dig> group mutants were also sensitive to killing by mms or bleomycin .
results of all survival tests are summarized in table  <dig>  in total,  <dig> of the  <dig> ecoris non-rad <dig> group mutants were sensitive to bleomycin  and  <dig> mutants were sensitive to mms .
surprisingly, growth of  <dig> strains was not affected by either mms or bleomycin.
the pronging assays are limited to detection of mutants that consistently exhibit ≥  <dig> fold fewer colonies than wildtype cells after exposure to dna damage.
it is possible that modest chemical sensitivities in some mutants could not be detected by this method.
a mutants were ranked as resistant , sensitive , or strongly sensitive .
mutants classified as ss exhibited > 25-fold higher killing than wildtype cells using semi-quantitative dilution pronging assays.
Γ, gamma radiation.
the results described above established that all  <dig> ecoris rad <dig> group mutants were sensitive to mms and bleomycin and that most of the  <dig> other ecoris mutants were also sensitive to one or both of the chemical clastogens.
survival of each of the mutants after exposure to a single dose of gamma radiation  was tested next using dilution pronging assays.
each of the rad <dig> group mutants was strongly sensitive to ionizing radiation but, unexpectedly, only  <dig> of the other  <dig> mutants showed cell killing at  <dig> krads .
each of these genes had been shown previously to affect radiation resistance in diploids  <cit> , but over half of them had no discernible impact on survival in the current haploid assays.
clastogen sensitivities for all  <dig> mutants are depicted schematically in figure  <dig>  the recombination-deficient rad <dig> group mutants  were sensitive to all  <dig> dsb-inducing agents, but only  <dig> of the remaining  <dig> mutants were similarly sensitive to all agents .
the genes inactivated in these  <dig> strains therefore affect sensitivity to enzymatic, chemical and physical dsb-generating agents, suggesting that they are most likely to affect dsb repair processes directly.
the  <dig> non-rad <dig> group mutants showed reduced survival when ecori was expressed and most of the strains were also sensitive to mms and bleomycin, but only  <dig> were killed by radiation.
differences in the methodologies employed for the assays may have contributed to this discrepancy.
in the current study, radiation resistance was determined after a single exposure of cells to gamma radiation lasting less than 60 min.
the cells were then incubated at 30°c on agar plates for several days, without further exposure, until the surviving cells grew into colonies and could be counted.
in the ecori, mms, and bleomycin experiments, however, cells were exposed continuously to these clastogens as they grew on agar plates for a similar number of days .
unlike the irradiated cells, these cells traversed multiple cell cycles while continuously exposed to dsbs.
cells are particularly sensitive to some types of dna damage in specific phases of the cell cycle, especially during s phase and mitosis  <cit> .
the different result obtained with radiation suggested the possibility that some genes were needed for survival when damage was constantly generated, but not in cells that experienced damage only one time in a single phase of the cycle.
bleomycin damages dna directly by entering cells, forming a free radical complex, and binding and cleaving dna in the nucleus.
cells can be exposed to this clastogen continuously by adding it to liquid or plate cell growth media.
it is also possible to perform a single exposure experiment by adding bleomycin to cells for a brief time, e.g., 30 min, and then washing the cells several times to remove the drug before spreading onto plates to determine survival.
most of the new mutants identified here were killed by continuous exposure to bleomycin in plates, but not by a single brief exposure to gamma radiation, which also generates dsbs primarily by free radical mechanisms  <cit> .
we hypothesized that these mutants would be similarly resistant to a brief exposure to bleomycin if continuous exposure was the key to their sensitivity.
to assess survival after a single exposure to bleomycin, cells were harvested from patches on plates, inoculated into ypda broth and grown to log phase at 30°c, followed by exposure to  <dig> μg/ml bleomycin for 30 min .
cells were washed, 10-fold serial dilutions were made and appropriate volumes of the diluted cultures were spread onto ypda plates and incubated at 30°c for three to four days to detect surviving colonies.
survival was strongly reduced in control rad <dig> cells and moderately reduced in rad <dig> mutants, which is in accord with their previously characterized phenotypes as part of the rad <dig> group .
five non-rad <dig> group mutants that were resistant to a brief exposure to gamma  were tested for their sensitivities to brief treatment with bleomycin.
survival of the gamma-resistant mutants was variable, ranging from near-wildtype in taf <dig> mutants to a modest 100-fold reduction in ctf <dig> mutants .
we then reasoned that there might be a different correlation: the gamma resistant mutants may simply exhibit a range of bleomycin sensitivities that is different than the range of sensitivities found among gamma sensitive mutants.
however, the five gamma sensitive mutants tested  showed sensitivities that were similar to the gamma resistant strains, with survival reduced from  <dig>  fold to  <dig>  fold .
the median fold reduction for the gamma sensitive mutants was only moderately larger than that of the gamma resistant strains .
these experiments indicate that there is not a simple correlation between sensitivities to a single brief exposure to bleomycin and to gamma radiation.
they also suggest that the small number of gamma sensitive mutants relative to ecori, mms and bleomycin sensitive mutants is unlikely to be due simply to different treatment methods involving continuous versus single-hit exposures.
averages of four trials and standard deviations are shown.
the saccharomyces genome database  contains detailed descriptions of all known and putative genes within the saccharomyces cerevisiae genome.
this resource was utilized to investigate the functions and potential relationships among the genes affecting survival after induction of ecori expression.
analysis of the chromosomal locations of each of the genes revealed that overlapping of two putative open reading frames  was a characteristic of  <dig> of the loci.
when overlapping occurs, it is not completely certain that loss of the gene that was precisely deleted in the library mutant  has caused the observed phenotype.
examples are shown in figure  <dig> , illustrating four different orientations of overlapping genes.
most of the overlapping orfs are predicted to be transcribed in different directions, but in some cases each orf was oriented in the same direction.
in most cases, one of the orfs has been shown experimentally to produce a protein in vivo, but transcription/translation of the other putative gene has not yet been established.
orfs that were specifically deleted in the library strains used for this project are shown in figure 4e  along with the overlapping orfs that were partially deleted .
at only one locus, containing mms <dig> and ybr099c, were both orfs separately deleted during the construction of the haploid strain library.
thus, both of these genes are listed in the left column of figure 4e.
at six of the loci, the gene that was precisely deleted has previously been shown to produce a protein product .
at most of the loci the orf that was deleted is unconfirmed and overlaps a verified gene.
this suggests that some mutants analyzed here  may exhibit phenotypes because of partial deletions of overlapping verified genes, not because of the orf whose coding region was deleted.
to investigate this possibility, matα library mutants containing precise deletions of the coding regions of  <dig> of the verified overlapping genes  were tested for sensitivity to ecori.
two of the strains, npl <dig> and rpl <dig>  were strongly sensitive to ecori expression , suggesting that inactivation/truncation of these verified genes may have been the actual cause of the ecoris phenotype in the original mutants.
growth of the remaining  <dig> mutants was not affected by ecori expression.
the source of the sensitivities of the original mutants with these latter  <dig> genes inactivated remains unclear.
it may be due to inactivation of the unverified genes  or it may be caused by production of truncated proteins from the partially deleted verified genes that produce phenotypes not seen when the genes are completely inactivated.
surprisingly, some of the  <dig> genes required for resistance to ecori were found to lie directly adjacent to each other.
when two genes are adjacent, deletion of one gene has the potential to exert polar effects on the transcription and/or mrna stability of the nearby gene.
at two loci on chromosomes x and xiii, the hsp <dig> and cis <dig> genes and the ubp <dig> and mre <dig> genes were found to be only 575 bp and 445 bp from each other, respectively .
at another region on chromosome xiii, the coding regions of trm <dig> and ubx <dig> are remarkably close , with the intergenic segment encompassing the entire presumptive promoter region of ubx <dig> .
the locus summary gene descriptions and gene ontology  annotations indicating biological processes compiled at the saccharomyces genome database were employed to analyze and sort the non-rad <dig> group genes.
for completeness, the analysis included verified genes that were partially deleted when an unverified gene was precisely deleted during creation of the library.
as shown in table  <dig>   <dig> of the non-rad <dig> group genes could be classified into groups with shared functions.
a large number  have previously been linked to transcription regulation.
many others are known to be involved in dna metabolism, affecting sister chromatid cohesion, histone and chromatin structure, nuclease processing of dna and chromosome segregation.
thus, most of the genes have functions that could potentially affect dna repair.
several genes have known roles in dna replication or repair, including exo <dig>  sae <dig>  rad <dig>  mms <dig>  and mms <dig> <cit> .
several other genes encode mitochondrial proteins, which may be an indication that repair of this genome is compromised in some mutants, though other explanations are possible.
a gene names separated by a forward slash indicate deletions within two overlapping open reading frames.
functions and processes are described for the verified gene only.
source: the saccharomyces genome database.
b the rtt <dig> gene is frequently referred to as rem <dig> in the literature.
high-throughput studies using yeast 2-hybrid screens, affinity capture coupled with mass spectrometry and other physical methods have pointed to extensive protein:protein interactions among the eight rad <dig> group proteins that are required for resistance to ecori .
each protein physically interacts with one or more other proteins in the rad <dig> group and also with other proteins involved in replication and repair.
interestingly, the proteomics studies have also provided evidence that many of the proteins encoded by the non-rad <dig> group genes interact with each other and with known dna repair proteins.
forty-one of the non-rad <dig> group proteins display such interactions, in some cases involving many different proteins .
for example, gcn <dig> is proposed to interact with  <dig> of the other non-rad <dig> group proteins and also with rad <dig>  providing a link to dsb repair pathways.
names of interacting proteins that affect ecori resistance are underlined in the table.
* includes proteins predicted to interact physically plus proteins that are shared components of a multisubunit complex.
underlined names indicate proteins encoded by genes that are required for resistance to ecori overexpression.
other interacting proteins have previously been linked to dsb repair and/or damage-inducible checkpoints.
associations were taken from the saccharomyces genome database.
many yeast proteins are conserved in lower and higher eukaryotes.
to examine conservation of the genes identified in this work, homologies of the protein products of the  <dig> ecoris loci were compared to proteins from three actively studied genomes of higher eukaryotes: human, mouse, and rat .
the analysis was conducted using the blastp sequence alignment program at the national center for biotechnology information , which produces homology scores that are sorted based on “e-values”.
scores with higher negative exponents, e.g., e- <dig>  indicate greater sequence homology, while smaller exponents indicate weak or no homology.
analysis of the sequences of the eight yeast rad <dig> group proteins revealed that seven of the polypeptides have strong homology with mammalian proteins, displaying e-values as high as e- <dig>  with the exception of xrs <dig>  scores ranged from e- <dig> to e- <dig>  xrs <dig> is known to be an ortholog of the human protein nbs <dig>  but its sequence shows only weak similarity to nbs <dig> in short regions at the amino and carboxy termini  <cit> .
forty-five of the other proteins exhibited moderate or strong homology  to proteins from all three mammalian genomes.
strongest homologies were seen with gnd <dig>  top <dig> and atp <dig>  which displayed scores of e- <dig>  e- <dig> and e- <dig>  respectively, compared to their human counterparts.
gnd <dig> regenerates nadph inside cells, top <dig> is a topoisomerase needed during transcription and dna replication, and atp <dig> is a component of a membrane complex important for synthesis of atp.
each of these reactions corresponds to an essential process common to all organisms and likely explains the unusually high conservation of the proteins.
fifty-two of the  <dig> proteins  displayed homology scores of e- <dig> or better, corresponding to 64% of the total.
this suggests that most of the yeast genes characterized here have functional counterparts in mammalian cells that are also likely to affect dna repair processes.
discussion
in this study, genes shown in previous genomic screens to be required for resistance to gamma radiation in diploid cells were analyzed to identify those genes that specifically affect repair of site-specific dsbs.
this was accomplished by expressing the restriction endonuclease ecori in haploid equivalents of each mutant and assessing survival after induction in galactose.
eighty-one mutants were identified as sensitive to ecori expression, including eight known rad <dig> group mutants plus seventy-three others.
a few genes were only tested in mata cells because the matα library versions grew too poorly to be evaluated.
this observation, in conjunction with others such as our finding that one of the matα strains  was ura+, indicate that a small fraction of the haploid library mutants contain uncharacterized mutations in other genes.
such secondary mutations can confound results of genome-wide screens, a concern that has been noted before  <cit> .
sensitivity to ecori expression was confirmed in both matα and mata strains for most mutants in the current study, however, indicating that the results observed using one haploid library were largely reproducible in the other library.
among rad <dig> group mutants, rad <dig>  rad <dig>  rad <dig>  rad <dig>  rad <dig>  rad <dig>  mre <dig> and xrs <dig> strains were ecoris, but rad <dig>  rdh <dig> and rad <dig> cells were not.
these results, in conjunction with our observation that nhej mutants such as yku <dig>  dnl <dig> and nej <dig> cells were not sensitive, indicate that homologous recombination is the most critical pathway in the s288c strain background used to construct the libraries.
the resistance of rad <dig>  rdh <dig> and rad <dig> cells is consistent with the more specialized roles of rad <dig> and rdh <dig> in recombination and the possible major function of rad <dig> in sister chromatid cohesion  <cit> .
the eight rad <dig> group mutants were sensitive to every clastogen tested, including bleomycin, mms and gamma radiation.
among the other  <dig> mutants,  <dig> exhibited reduced survival after exposure to bleomycin and  <dig> mutants were sensitive to mms.
the pronging survival assays employed here were limited to detection of mutants that showed a consistent reduction of  <dig> fold or more in colony formation.
thus, modest sensitivities in some mutants may not have been detectable.
although each of the eight rad <dig> group mutants was sensitive to gamma radiation as expected, a surprisingly small fraction of the other ecoris mutants exhibited this phenotype .
thus, each of the non-rad <dig> group mutants was found to be gamma-sensitive as diploids in previous genomic screens  <cit> , but most of them were not detectably sensitive as haploids in the current study.
in the previous study by bennett et al., diploid mutants were categorized as gamma sensitive if there was  reduced survival in pronging assays or  a slow recovery from gamma-induced damage  even if the number of surviving colonies was similar to wt cells  <cit> .
in the screen by game et al.,  <cit> , a very different method was utilized involving irradiating pools of diploid deletion library strains, purifying and amplifying dna from the surviving cells, and using hybridization methods to identify mutants that had reduced abundance in the culture due to cell killing.
this latter study detected  <dig> strongly radiation-sensitive mutants,  <dig> of which were also identified in the report by bennett et al.
in the current study using haploid cells, mutants were only scored as sensitive in semi-quantitative pronging assays if survival was reduced, i.e., the number of surviving colonies on plates was reproducibly decreased by ≥ 2-fold.
these differences in methodology and classification are likely to explain many of the differences in radiation sensitivities observed between haploid and diploid strains.
however, in at least some cases the differences may be a characteristic of the strains: srs <dig> and rdh <dig> mutants are known to be radiation sensitive as diploids, but not as haploid cells, and other mutants may share this phenotype  <cit> .
seventeen of the ecoris mutants contained deletions of an orf that overlapped one or more other orfs.
at only one locus, containing the divergent and overlapping genes mms <dig> and ybr099c, did the library collection contain separate mutants containing precise deletions of each overlapping orf.
among the  <dig> other loci,  <dig> verified genes that were completely deleted in the library strains  overlapped unverified orfs and  <dig> unverified orfs whose coding regions were deleted overlapped verified genes .
many of the unverified orfs are likely to represent nonfunctional genes, so at most loci only one of the overlapping genes is transcribed and translated in vivo.
however, it is possible that both genes are functional at some of the overlapping regions: a search of the sequences stored at the saccharomyces genome database revealed that  <dig> yeast chromosomal loci contain overlapping protein-coding genes that are each verified  and a region on chromosome v contains  <dig> verified genes whose coding regions overlap each other .
among the ten mutants containing a deleted unverified orf that overlapped a verified gene, it was possible that the sensitivity phenotypes were not caused by the loss of the putative gene that was precisely deleted to create the library strain.
instead, truncation of the overlapping gene could have generated the sensitivity.
tests using seven mutants containing precise deletions of the coding regions of overlapping verified genes revealed that two of them, rpl <dig> and npl <dig>  were strongly ecoris, and five others were not.
it is therefore possible that the phenotypes of bud <dig> and ydr433w deletion mutants are actually caused by inactivation of the overlapping rpl <dig> and npl <dig> genes, respectively.
additional work will be required to confirm this conjecture and to determine the source of the dna damage sensitivities in the other mutants containing deletions affecting two or more overlapping orfs.
forty-one of the non-rad <dig> group genes have previously been linked to nuclear processes such as transcription, nuclease processing of dna, histone modification, chromosome segregation and sister chromatid cohesion.
some of the genes are already known to affect repair of dsbs.
these include exo <dig> and sae <dig>  involved in nuclease resection of dsbs during homologous recombination, and mms <dig>  which encodes a subunit of the mms4-mus <dig> endonuclease involved in cleavage of branched dna structures  <cit> .
detection of mutants affecting mitochondria-associated proteins suggests that repair of this organelle’s genome may be compromised in some mutants.
six of the  <dig> mitochondrial mutants were also sensitive to mms and/or bleomycin, supporting the idea that a defect in repair of damage to dna is the main cause of sensitivity in the mutants.
recent studies have demonstrated that many dna and rna processing enzymes utilize iron-sulfur clusters, which are synthesized primarily in the mitochondria, indicating a possible source of the sensitivity  <cit> .
the potential roles of several of the other genes in dna repair, such as those known to affect cell membrane architecture, remains unclear.
rad <dig> group proteins exhibit physical interactions with each other and with many other proteins involved in dna replication and repair .
forty-one of the non-rad <dig> group proteins have previously been shown to interact with at least one other protein affecting dna damage resistance.
some proteins, such as ddc <dig> and gcn <dig>  exhibit multiple associations with both rad <dig> group proteins and with other repair proteins.
these linkages to known repair proteins imply that some of the proteins may have direct roles in dna repair.
our observation that 64% of the proteins exhibit moderate or strong homology to mammalian proteins suggests further that many of the functions identified in yeast cells will be conserved in higher eukaryotes.
investigation of the clastogen sensitivities of the mutants characterized in this study revealed unexpected variability.
for example, among the  <dig> non-rad <dig> group mutants that were ecoris, only  <dig> strains were sensitive to all three other clastogens: mms, bleomycin and gamma radiation.
the remaining strains exhibited mixed sensitivity phenotypes .
most of the mutants were sensitive to mms and bleomycin, but only  <dig> were killed by gamma radiation.
since the radiation survival studies involved a single brief exposure and the other three damaging treatments were applied continuously during colony formation on plates, it was possible that the gamma-resistant mutants were only sensitive to agents that generate damage continuously throughout the cell cycle.
to test this idea, survival of gamma-resistant and gamma-sensitive mutants was tested after a single brief exposure to the direct dna-damaging agent bleomycin.
all of the radiation-resistant and radiation-sensitive mutants displayed similar, modest sensitivities to a brief exposure to bleomycin, suggesting that continous exposure was not the key to their sensitivities.
although the cells were exposed to bleomycin for a short time  and were washed extensively afterward, it is possible that some of the drug persisted inside the cells for more than one cell cycle.
such persistence would increase the difficulty of making comparisons between effects caused by radiation versus chemical agents.
all of the mutants characterized in this study have been shown to be radiation-sensitive as diploids  and each mutant was also sensitive to in vivo expression of ecori, an endonuclease that specifically induces site-specific, cohesive-ended dsbs in dna.
however, over half of the non-rad <dig> group mutants were not radiation sensitive as haploid cells.
furthermore, the haploid strains exhibited variable sensitivities to the chemical clastogens mms and bleomycin .
this phenotypic variation, in conjunction with variability in sensitivities seen in other studies assessing the effects of multiple chemicals on yeast cell growth e.g., ref.
<cit> , indicate that caution is needed in interpretation of such experiments.
CONCLUSIONS
in this study a large number of genes were identified that are essential for survival of haploid yeast cells when ecori endonuclease is expressed in vivo.
mutant phenotypes observed in strains of one haploid library were largely confirmed upon testing of the equivalent mutants from another library.
many library mutants contained deletions of a single putative gene that truncated another overlapping gene; experiments indicated that phenotypes observed in such mutants could be caused, in some cases, by alteration of the overlapping gene.
many of the genes affecting ecori sensitivity have previously been linked to dna and rna metabolism and several are active in processes known to affect dsb repair, including nucleosome remodeling, sister chromatid cohesion, and dna damage responsive cell cycle checkpoints.
determining which of the genes are directly involved in dsb repair pathways will require application of specific in vivo assays for recombination and nhej and direct physical measurements of dsb repair.

 chromosomal dna within eukaryotic cells is constantly subjected to many types of damage from both endogenous and exogenous sources. repair of the damage may be performed by one or more different pathways, depending on the specific type of dna lesion involved. major pathways that have been characterized include nucleotide excision repair, active on ultraviolet light induced dna crosslinks and other bulky lesions, base excision repair for repair of damaged and lost bases, and mismatch repair. two other pathways, homologous recombination and nonhomologous end-joining, are active in repair of dna double-strand breaks. dsbs can be induced by endogenous processes such as oxidation, arrest of dna replication forks, nuclease cleavage or processing of various dna lesions and by exposure to external agents such as ionizing radiation or clastogenic chemicals. dsb ends may be blunt or may contain single-strand overhangs. the ends may also have other associated damage such as altered or missing bases, e.g., as seen after cells are exposed to ionizing radiation or the chemical bleomycin. most dna damaging agents that induce dsbs generate other lesions as well, with the other lesions typically occurring at much higher frequencies. despite their rarity, unrepaired and/or misrepaired dsbs are believed to be the major cause of cell death after exposure to such agents. repair of dsbs by nhej and hr involves the concerted actions of many conserved proteins found in both single-celled and multicellular organisms. in saccharomyces cerevisiae, or budding yeast, nhej requires at least three factors. the yku70/yku complex binds to dna ends, provides protection from nucleases, and recruits other repair proteins. the mre11/rad50/xrs complex forms a bridge between the broken dna ends and also has nuclease activities involved in processing of some end structures. dnl4/lif1/nej represents the dna ligase iv complex, which covalently links broken dna strands together. nej may also function earlier in the pathway, modulating the binding of yku70/yku to dna ends. the chromatin silencing proteins sir sir and sir are also required in yeast cells, but they primarily affect nhej indirectly through their effects on nej gene expression. other proteins such as exo rad pol the rsc and smc complexes, and dna damage checkpoint proteins have also been suggested to modulate the efficiency of nhej repair. repair by the homologous recombination pathway involves association of broken dsb ends with an unbroken homologous dna molecule such as a sister chromatid or a homologous chromosome, which then serves as a template for repair. efficient repair by this pathway requires the functions of many proteins, especially several members of the rad group. this group of genes was originally defined in the 1970s on the basis of mutant phenotypes. with the exceptions of rad and rdh which are weak homologues of rad and rad respectively, mutants with defects in these genes share many common phenotypes. they exhibit strong sensitivity to ionizing radiation and chemical clastogens, weak sensitivity to ultraviolet light, and genetic epistasis when combined with mutations in other members of the group to generate double mutants. two other original members of this group are rad now known to function primarily in the dna damage-induced checkpoint response, and rad a locus that remains uncharacterized. an additional gene, rad has also been suggested as a member, though its main function may lie in sister chromatid cohesion. the general model for recombinational repair of dsbs involves several steps. first, broken ends are resected by mrx and other enzymes such as exo sgs sae and dna to create long 3’-ended single-stranded dna tails. the tails are subsequently bound by the ssdna binding protein complex, rad and other proteins that recruit factors promoting a homology search, strand invasion of an unbroken dna molecule, new dna synthesis and other steps ultimately leading to repair of the broken strands and separation of the paired complexes. several additional proteins have been shown to influence the efficiency of the pathway. these include components of the dna damage checkpoint response, nucleosome remodeling, sister chromatid cohesion, other end-processing nucleases, resolvase-like enzymes and chromatin reassembly proteins. the development of yeast deletion strain libraries has allowed researchers to screen thousands of individual gene mutants in a single experiment to identify all mutants that have a particular phenotype. several previous studies have involved testing such strains for sensitivities to physical or chemical agents that damage dna, including ionizing radiation, ultraviolet light, methyl methanesulfonate and other chemicals. genomic screens by bennett et al. and game et al. using over diploid mutants identified a large number of genes that are required for resistance to gamma radiation. ionizing radiation generates low levels of dsbs in cellular dna, but it induces higher frequencies of many other types of dna damage and also causes damage to other macromolecules inside the cell. thus, only a subset of the genes found to be required for radiation resistance are actually likely to affect dsb repair. the bacterial restriction endonuclease ecori recognizes and cuts the palindromic 6 bp sequence g^aattc, generating 5’ ssdna overhangs that are nt long. controlled in vivo expression of this nuclease from a galactose-regulated promoter has been employed in several studies investigating cellular responses to dsbs and their repair. use of ecori was advantageous for these studies because it is believed to produce essentially only one type of dna lesion, a dsb. thus, it is more specific than commonly used clastogens like radiation, mms or bleomycin. ecori-induced dsbs are repaired efficiently in most wildtype haploid strains of yeast, but produce strong growth inhibition and modest killing in most recombination-deficient rad group mutants. in contrast, the impact of ecori expression on nhej mutants is more variable and dependent on the strain background employed for the study. this characteristic is similar to the variable sensitivities to mms and bleomycin that have been observed in nhej mutants. progress in understanding dsb repair pathways has been hindered because many of the proteins acting on or influencing efficiency at each of the various steps have remained unidentified. in the current study we tested each of the genes shown previously to be important for resistance to ionizing radiation, using both matα and mata haploid mutant libraries, to identify those genes that specifically affect dsb repair. this goal was accomplished by screening the mutants for sensitivity to dsbs produced by in vivo expression of ecori. use of two libraries was advantageous because it allowed phenotypes observed in one haploid mutant to be confirmed in an equivalent mutant of opposite mating type. these efforts have resulted in the identification and characterization of non-rad group genes that are required for efficient repair of site-specific dsbs.  two previous screens undertaken using diploid yeast deletion strain libraries, identified a total of mutants with reduced resistance to gamma radiation. several of these mutants corresponded to known rad group genes defective in recombination, but most genes had not previously been associated with dna repair. because radiation generates many types of cellular damage, only a subset of these genes are likely to affect dsb repair. to identify genes specifically impacting repair of dsbs, haploid matα versions of each of the mutants were tested for resistance to dsbs produced by in vivo expression of ecori endonuclease from a gal promoter. one more library mutant, exo was added to the screen, bringing the total to exo is a 5’-to’3’ exonuclease active in resection of dsb ends in the homologous recombination pathway and it has other roles in replication and mismatch repair. although exo1−/− cells were not detected in the original diploid screens, we have previously observed that exo haploid library mutants are gamma-sensitive and human exo1−/− cells are sensitive to ionizing radiation. for these experiments, growth of cells containing the control vector prs or the gal1p::ecori fusion plasmid ycpgal::rib was initially analyzed by “double-imprint” replica-plating patches of cells from plates containing 2% raffinose to plates containing 2% galactose. mutants whose growth was inhibited in the presence of galactose were then retested using more quantitative dilution pronging survival assays. for each pronging assay, cells containing either the vector or the gal1p::ecori plasmid were harvested from patches on raffinose plates, serially diluted 5-fold into 96-well microtiter dishes, and pronged to plates containing raffinose or galactose. tests of control matα haploid library strains revealed that survival of wildtype by cells expressing ecori was high. in contrast, cell survival and growth rates were reduced in several rad group recombination mutants. colony formation in mutants defective in nhej repair was similar to that of wildtype cells in this strain. this phenotype was not unexpected because dna damage sensitivities of nhej mutants are known to vary in different strain backgrounds. testing of the haploid matα library mutants revealed that survival of many of the strains was reduced by expression of ecori. results obtained with two mutants, mms and mms are depicted in figure 1b along with wt and mre strain controls. interestingly, of the mutants were successfully evaluated using the procedure described above, but of the matα library strains could not be tested, usually because they grew too poorly on galactose plates. some mutants could not be tested for other reasons. ade mutants did not produce ura+ colonies even after repeated transformations with prs and ycpgal:rib plasmid dnas. also, cdc cells from the library were phenotypically ura+, preventing use of the ura selectable marker on the two plasmids. a total of of the matα mutants that could be tested exhibited killing when ecori was expressed. to address the matα library strains that were untestable, haploid mutants with the equivalent genes inactivated were obtained from an alternative deletion strain collection of opposite mating type. twenty-two of these mata strains grew on galactose media and could be successfully transformed with the assay plasmids. this result suggests that many of the original matα library versions of the strains had secondary mutations affecting carbohydrate metabolism. only two of the mata mutants, ade and hfi remained untestable. similar to the matα library strains, transformations of mata ade mutants did not produce ura+ colonies, and hfi cells from both libraries grew too poorly to be tested. eleven of the mata mutants were found to be ecori-sensitive. thus, of the original genes were tested, as matα strains and as mata cells, and of them were identified as important for normal resistance to ecori-induced dsbs in haploid cells. as shown in the top section of table  eight members of the rad group were required for normal resistance to ecori. deletions of two other group members, rad and rdh had no detectable effect. another gene proposed to be a member of the group called rad was also not required for repair of the ecori-induced dsbs. both mata and matα versions of rad rdh and rad strains were tested and found to be resistant to ecori. all mutants in table  were classified as moderately sensitive or strongly sensitive, based on the extent of colony formation seen in the semi-quantitative pronging survival assays. a eight rad group mutants are shown at the top of the table. mutants were ranked as resistant, sensitive, or strongly sensitive. n/d usually indicates that the library strain was unable to grow on galactose plates. some genes could not be tested for other reasons, e.g., matα cdc cells were ura+. b gene names separated by a forward slash indicate deletions affecting two or more overlapping open reading frames. the coding region of the gene listed first was deleted in each strain. c the matα mre strain was reconstructed for this study because the library mutant did not exhibit phenotypes characteristic of mre mutants. drtt is usually referred to as rem in the literature. eyml009w-b overlaps the verified gene spt and another open reading frame called yml009c-a. because of the apparent high frequency of secondary mutations in the original library, experiments were performed to determine if ecori sensitivities seen in the matα mutants could be confirmed in mata versions of the strains. the ecoris phenotype was reproduced in of the equivalent mata strains using dilution pronging as before, with only psy rad and spt mata library strains exhibiting resistance. these experiments therefore identified rad group mutants and non-rad group mutants as important for survival after induction of ecori. for of the latter mutants, sensitivity was confirmed in both matα and mata strains. for completeness, all of the library mutants that were tested in this study and found to be resistant to ecori are listed in additional file 1: table s the mutants exhibiting sensitivity to ecori were further characterized by assessing their survival after exposure to the chemicals mms and bleomycin. these chemicals induce dsbs by very different mechanisms and have been widely used to investigate dna repair pathways. the eight rad group mutants plus all other mutants were analyzed using dilution pronging survival assays. strains from the original matα library were used for the experiments, with mata library strains substituted only for matα cells that had growth defects. rad group mutants exhibited strong killing on plates containing either 2 mm mms or 4 μg/ml bleomycin. rad and rad mutants were least sensitive in the assays, which were conducted at 30°c; these mutants exhibit greatest sensitivity to dna damaging agents at 23°c. higher concentrations of mms and bleomycin were not used because they caused strong growth inhibition of wt cells. many of the non-rad group mutants were also sensitive to killing by mms or bleomycin. results of all survival tests are summarized in table  in total, of the ecoris non-rad group mutants were sensitive to bleomycin and mutants were sensitive to mms. surprisingly, growth of strains was not affected by either mms or bleomycin. the pronging assays are limited to detection of mutants that consistently exhibit ≥  fold fewer colonies than wildtype cells after exposure to dna damage. it is possible that modest chemical sensitivities in some mutants could not be detected by this method. a mutants were ranked as resistant, sensitive, or strongly sensitive. mutants classified as ss exhibited > 25-fold higher killing than wildtype cells using semi-quantitative dilution pronging assays. Γ, gamma radiation. the results described above established that all ecoris rad group mutants were sensitive to mms and bleomycin and that most of the other ecoris mutants were also sensitive to one or both of the chemical clastogens. survival of each of the mutants after exposure to a single dose of gamma radiation was tested next using dilution pronging assays. each of the rad group mutants was strongly sensitive to ionizing radiation but, unexpectedly, only of the other mutants showed cell killing at krads. each of these genes had been shown previously to affect radiation resistance in diploids, but over half of them had no discernible impact on survival in the current haploid assays. clastogen sensitivities for all mutants are depicted schematically in figure  the recombination-deficient rad group mutants were sensitive to all dsb-inducing agents, but only of the remaining mutants were similarly sensitive to all agents. the genes inactivated in these strains therefore affect sensitivity to enzymatic, chemical and physical dsb-generating agents, suggesting that they are most likely to affect dsb repair processes directly. the non-rad group mutants showed reduced survival when ecori was expressed and most of the strains were also sensitive to mms and bleomycin, but only were killed by radiation. differences in the methodologies employed for the assays may have contributed to this discrepancy. in the current study, radiation resistance was determined after a single exposure of cells to gamma radiation lasting less than 60 min. the cells were then incubated at 30°c on agar plates for several days, without further exposure, until the surviving cells grew into colonies and could be counted. in the ecori, mms, and bleomycin experiments, however, cells were exposed continuously to these clastogens as they grew on agar plates for a similar number of days. unlike the irradiated cells, these cells traversed multiple cell cycles while continuously exposed to dsbs. cells are particularly sensitive to some types of dna damage in specific phases of the cell cycle, especially during s phase and mitosis. the different result obtained with radiation suggested the possibility that some genes were needed for survival when damage was constantly generated, but not in cells that experienced damage only one time in a single phase of the cycle. bleomycin damages dna directly by entering cells, forming a free radical complex, and binding and cleaving dna in the nucleus. cells can be exposed to this clastogen continuously by adding it to liquid or plate cell growth media. it is also possible to perform a single exposure experiment by adding bleomycin to cells for a brief time, e.g., 30 min, and then washing the cells several times to remove the drug before spreading onto plates to determine survival. most of the new mutants identified here were killed by continuous exposure to bleomycin in plates, but not by a single brief exposure to gamma radiation, which also generates dsbs primarily by free radical mechanisms. we hypothesized that these mutants would be similarly resistant to a brief exposure to bleomycin if continuous exposure was the key to their sensitivity. to assess survival after a single exposure to bleomycin, cells were harvested from patches on plates, inoculated into ypda broth and grown to log phase at 30°c, followed by exposure to μg/ml bleomycin for 30 min. cells were washed, 10-fold serial dilutions were made and appropriate volumes of the diluted cultures were spread onto ypda plates and incubated at 30°c for three to four days to detect surviving colonies. survival was strongly reduced in control rad cells and moderately reduced in rad mutants, which is in accord with their previously characterized phenotypes as part of the rad group. five non-rad group mutants that were resistant to a brief exposure to gamma were tested for their sensitivities to brief treatment with bleomycin. survival of the gamma-resistant mutants was variable, ranging from near-wildtype in taf mutants to a modest 100-fold reduction in ctf mutants. we then reasoned that there might be a different correlation: the gamma resistant mutants may simply exhibit a range of bleomycin sensitivities that is different than the range of sensitivities found among gamma sensitive mutants. however, the five gamma sensitive mutants tested showed sensitivities that were similar to the gamma resistant strains, with survival reduced from fold to fold. the median fold reduction for the gamma sensitive mutants was only moderately larger than that of the gamma resistant strains. these experiments indicate that there is not a simple correlation between sensitivities to a single brief exposure to bleomycin and to gamma radiation. they also suggest that the small number of gamma sensitive mutants relative to ecori, mms and bleomycin sensitive mutants is unlikely to be due simply to different treatment methods involving continuous versus single-hit exposures. averages of four trials and standard deviations are shown. the saccharomyces genome database contains detailed descriptions of all known and putative genes within the saccharomyces cerevisiae genome. this resource was utilized to investigate the functions and potential relationships among the genes affecting survival after induction of ecori expression. analysis of the chromosomal locations of each of the genes revealed that overlapping of two putative open reading frames was a characteristic of of the loci. when overlapping occurs, it is not completely certain that loss of the gene that was precisely deleted in the library mutant has caused the observed phenotype. examples are shown in figure , illustrating four different orientations of overlapping genes. most of the overlapping orfs are predicted to be transcribed in different directions, but in some cases each orf was oriented in the same direction. in most cases, one of the orfs has been shown experimentally to produce a protein in vivo, but transcription/translation of the other putative gene has not yet been established. orfs that were specifically deleted in the library strains used for this project are shown in figure 4e along with the overlapping orfs that were partially deleted. at only one locus, containing mms and ybr099c, were both orfs separately deleted during the construction of the haploid strain library. thus, both of these genes are listed in the left column of figure 4e. at six of the loci, the gene that was precisely deleted has previously been shown to produce a protein product. at most of the loci the orf that was deleted is unconfirmed and overlaps a verified gene. this suggests that some mutants analyzed here may exhibit phenotypes because of partial deletions of overlapping verified genes, not because of the orf whose coding region was deleted. to investigate this possibility, matα library mutants containing precise deletions of the coding regions of of the verified overlapping genes were tested for sensitivity to ecori. two of the strains, npl and rpl were strongly sensitive to ecori expression, suggesting that inactivation/truncation of these verified genes may have been the actual cause of the ecoris phenotype in the original mutants. growth of the remaining mutants was not affected by ecori expression. the source of the sensitivities of the original mutants with these latter genes inactivated remains unclear. it may be due to inactivation of the unverified genes or it may be caused by production of truncated proteins from the partially deleted verified genes that produce phenotypes not seen when the genes are completely inactivated. surprisingly, some of the genes required for resistance to ecori were found to lie directly adjacent to each other. when two genes are adjacent, deletion of one gene has the potential to exert polar effects on the transcription and/or mrna stability of the nearby gene. at two loci on chromosomes x and xiii, the hsp and cis genes and the ubp and mre genes were found to be only 575 bp and 445 bp from each other, respectively. at another region on chromosome xiii, the coding regions of trm and ubx are remarkably close, with the intergenic segment encompassing the entire presumptive promoter region of ubx. the locus summary gene descriptions and gene ontology annotations indicating biological processes compiled at the saccharomyces genome database were employed to analyze and sort the non-rad group genes. for completeness, the analysis included verified genes that were partially deleted when an unverified gene was precisely deleted during creation of the library. as shown in table  of the non-rad group genes could be classified into groups with shared functions. a large number have previously been linked to transcription regulation. many others are known to be involved in dna metabolism, affecting sister chromatid cohesion, histone and chromatin structure, nuclease processing of dna and chromosome segregation. thus, most of the genes have functions that could potentially affect dna repair. several genes have known roles in dna replication or repair, including exo sae rad mms and mms. several other genes encode mitochondrial proteins, which may be an indication that repair of this genome is compromised in some mutants, though other explanations are possible. a gene names separated by a forward slash indicate deletions within two overlapping open reading frames. functions and processes are described for the verified gene only. source: the saccharomyces genome database. b the rtt gene is frequently referred to as rem in the literature. high-throughput studies using yeast 2-hybrid screens, affinity capture coupled with mass spectrometry and other physical methods have pointed to extensive protein:protein interactions among the eight rad group proteins that are required for resistance to ecori. each protein physically interacts with one or more other proteins in the rad group and also with other proteins involved in replication and repair. interestingly, the proteomics studies have also provided evidence that many of the proteins encoded by the non-rad group genes interact with each other and with known dna repair proteins. forty-one of the non-rad group proteins display such interactions, in some cases involving many different proteins. for example, gcn is proposed to interact with of the other non-rad group proteins and also with rad providing a link to dsb repair pathways. names of interacting proteins that affect ecori resistance are underlined in the table. * includes proteins predicted to interact physically plus proteins that are shared components of a multisubunit complex. underlined names indicate proteins encoded by genes that are required for resistance to ecori overexpression. other interacting proteins have previously been linked to dsb repair and/or damage-inducible checkpoints. associations were taken from the saccharomyces genome database. many yeast proteins are conserved in lower and higher eukaryotes. to examine conservation of the genes identified in this work, homologies of the protein products of the ecoris loci were compared to proteins from three actively studied genomes of higher eukaryotes: human, mouse, and rat. the analysis was conducted using the blastp sequence alignment program at the national center for biotechnology information, which produces homology scores that are sorted based on “e-values”. scores with higher negative exponents, e.g., e- indicate greater sequence homology, while smaller exponents indicate weak or no homology. analysis of the sequences of the eight yeast rad group proteins revealed that seven of the polypeptides have strong homology with mammalian proteins, displaying e-values as high as e- with the exception of xrs scores ranged from e- to e- xrs is known to be an ortholog of the human protein nbs but its sequence shows only weak similarity to nbs in short regions at the amino and carboxy termini. forty-five of the other proteins exhibited moderate or strong homology to proteins from all three mammalian genomes. strongest homologies were seen with gnd top and atp which displayed scores of e- e- and e- respectively, compared to their human counterparts. gnd regenerates nadph inside cells, top is a topoisomerase needed during transcription and dna replication, and atp is a component of a membrane complex important for synthesis of atp. each of these reactions corresponds to an essential process common to all organisms and likely explains the unusually high conservation of the proteins. fifty-two of the proteins displayed homology scores of e- or better, corresponding to 64% of the total. this suggests that most of the yeast genes characterized here have functional counterparts in mammalian cells that are also likely to affect dna repair processes. discussion in this study, genes shown in previous genomic screens to be required for resistance to gamma radiation in diploid cells were analyzed to identify those genes that specifically affect repair of site-specific dsbs. this was accomplished by expressing the restriction endonuclease ecori in haploid equivalents of each mutant and assessing survival after induction in galactose. eighty-one mutants were identified as sensitive to ecori expression, including eight known rad group mutants plus seventy-three others. a few genes were only tested in mata cells because the matα library versions grew too poorly to be evaluated. this observation, in conjunction with others such as our finding that one of the matα strains was ura+, indicate that a small fraction of the haploid library mutants contain uncharacterized mutations in other genes. such secondary mutations can confound results of genome-wide screens, a concern that has been noted before. sensitivity to ecori expression was confirmed in both matα and mata strains for most mutants in the current study, however, indicating that the results observed using one haploid library were largely reproducible in the other library. among rad group mutants, rad rad rad rad rad rad mre and xrs strains were ecoris, but rad rdh and rad cells were not. these results, in conjunction with our observation that nhej mutants such as yku dnl and nej cells were not sensitive, indicate that homologous recombination is the most critical pathway in the s288c strain background used to construct the libraries. the resistance of rad rdh and rad cells is consistent with the more specialized roles of rad and rdh in recombination and the possible major function of rad in sister chromatid cohesion. the eight rad group mutants were sensitive to every clastogen tested, including bleomycin, mms and gamma radiation. among the other mutants, exhibited reduced survival after exposure to bleomycin and mutants were sensitive to mms. the pronging survival assays employed here were limited to detection of mutants that showed a consistent reduction of fold or more in colony formation. thus, modest sensitivities in some mutants may not have been detectable. although each of the eight rad group mutants was sensitive to gamma radiation as expected, a surprisingly small fraction of the other ecoris mutants exhibited this phenotype. thus, each of the non-rad group mutants was found to be gamma-sensitive as diploids in previous genomic screens, but most of them were not detectably sensitive as haploids in the current study. in the previous study by bennett et al., diploid mutants were categorized as gamma sensitive if there was reduced survival in pronging assays or a slow recovery from gamma-induced damage even if the number of surviving colonies was similar to wt cells. in the screen by game et al.,, a very different method was utilized involving irradiating pools of diploid deletion library strains, purifying and amplifying dna from the surviving cells, and using hybridization methods to identify mutants that had reduced abundance in the culture due to cell killing. this latter study detected strongly radiation-sensitive mutants, of which were also identified in the report by bennett et al. in the current study using haploid cells, mutants were only scored as sensitive in semi-quantitative pronging assays if survival was reduced, i.e., the number of surviving colonies on plates was reproducibly decreased by ≥ 2-fold. these differences in methodology and classification are likely to explain many of the differences in radiation sensitivities observed between haploid and diploid strains. however, in at least some cases the differences may be a characteristic of the strains: srs and rdh mutants are known to be radiation sensitive as diploids, but not as haploid cells, and other mutants may share this phenotype. seventeen of the ecoris mutants contained deletions of an orf that overlapped one or more other orfs. at only one locus, containing the divergent and overlapping genes mms and ybr099c, did the library collection contain separate mutants containing precise deletions of each overlapping orf. among the other loci, verified genes that were completely deleted in the library strains overlapped unverified orfs and unverified orfs whose coding regions were deleted overlapped verified genes. many of the unverified orfs are likely to represent nonfunctional genes, so at most loci only one of the overlapping genes is transcribed and translated in vivo. however, it is possible that both genes are functional at some of the overlapping regions: a search of the sequences stored at the saccharomyces genome database revealed that yeast chromosomal loci contain overlapping protein-coding genes that are each verified and a region on chromosome v contains verified genes whose coding regions overlap each other. among the ten mutants containing a deleted unverified orf that overlapped a verified gene, it was possible that the sensitivity phenotypes were not caused by the loss of the putative gene that was precisely deleted to create the library strain. instead, truncation of the overlapping gene could have generated the sensitivity. tests using seven mutants containing precise deletions of the coding regions of overlapping verified genes revealed that two of them, rpl and npl were strongly ecoris, and five others were not. it is therefore possible that the phenotypes of bud and ydr433w deletion mutants are actually caused by inactivation of the overlapping rpl and npl genes, respectively. additional work will be required to confirm this conjecture and to determine the source of the dna damage sensitivities in the other mutants containing deletions affecting two or more overlapping orfs. forty-one of the non-rad group genes have previously been linked to nuclear processes such as transcription, nuclease processing of dna, histone modification, chromosome segregation and sister chromatid cohesion. some of the genes are already known to affect repair of dsbs. these include exo and sae involved in nuclease resection of dsbs during homologous recombination, and mms which encodes a subunit of the mms4-mus endonuclease involved in cleavage of branched dna structures. detection of mutants affecting mitochondria-associated proteins suggests that repair of this organelle’s genome may be compromised in some mutants. six of the mitochondrial mutants were also sensitive to mms and/or bleomycin, supporting the idea that a defect in repair of damage to dna is the main cause of sensitivity in the mutants. recent studies have demonstrated that many dna and rna processing enzymes utilize iron-sulfur clusters, which are synthesized primarily in the mitochondria, indicating a possible source of the sensitivity. the potential roles of several of the other genes in dna repair, such as those known to affect cell membrane architecture, remains unclear. rad group proteins exhibit physical interactions with each other and with many other proteins involved in dna replication and repair. forty-one of the non-rad group proteins have previously been shown to interact with at least one other protein affecting dna damage resistance. some proteins, such as ddc and gcn exhibit multiple associations with both rad group proteins and with other repair proteins. these linkages to known repair proteins imply that some of the proteins may have direct roles in dna repair. our observation that 64% of the proteins exhibit moderate or strong homology to mammalian proteins suggests further that many of the functions identified in yeast cells will be conserved in higher eukaryotes. investigation of the clastogen sensitivities of the mutants characterized in this study revealed unexpected variability. for example, among the non-rad group mutants that were ecoris, only strains were sensitive to all three other clastogens: mms, bleomycin and gamma radiation. the remaining strains exhibited mixed sensitivity phenotypes. most of the mutants were sensitive to mms and bleomycin, but only were killed by gamma radiation. since the radiation survival studies involved a single brief exposure and the other three damaging treatments were applied continuously during colony formation on plates, it was possible that the gamma-resistant mutants were only sensitive to agents that generate damage continuously throughout the cell cycle. to test this idea, survival of gamma-resistant and gamma-sensitive mutants was tested after a single brief exposure to the direct dna-damaging agent bleomycin. all of the radiation-resistant and radiation-sensitive mutants displayed similar, modest sensitivities to a brief exposure to bleomycin, suggesting that continous exposure was not the key to their sensitivities. although the cells were exposed to bleomycin for a short time and were washed extensively afterward, it is possible that some of the drug persisted inside the cells for more than one cell cycle. such persistence would increase the difficulty of making comparisons between effects caused by radiation versus chemical agents. all of the mutants characterized in this study have been shown to be radiation-sensitive as diploids and each mutant was also sensitive to in vivo expression of ecori, an endonuclease that specifically induces site-specific, cohesive-ended dsbs in dna. however, over half of the non-rad group mutants were not radiation sensitive as haploid cells. furthermore, the haploid strains exhibited variable sensitivities to the chemical clastogens mms and bleomycin. this phenotypic variation, in conjunction with variability in sensitivities seen in other studies assessing the effects of multiple chemicals on yeast cell growth e.g., ref., indicate that caution is needed in interpretation of such experiments.  in this study a large number of genes were identified that are essential for survival of haploid yeast cells when ecori endonuclease is expressed in vivo. mutant phenotypes observed in strains of one haploid library were largely confirmed upon testing of the equivalent mutants from another library. many library mutants contained deletions of a single putative gene that truncated another overlapping gene; experiments indicated that phenotypes observed in such mutants could be caused, in some cases, by alteration of the overlapping gene. many of the genes affecting ecori sensitivity have previously been linked to dna and rna metabolism and several are active in processes known to affect dsb repair, including nucleosome remodeling, sister chromatid cohesion, and dna damage responsive cell cycle checkpoints. determining which of the genes are directly involved in dsb repair pathways will require application of specific in vivo assays for recombination and nhej and direct physical measurements of dsb repair. 
dsb repair is mediated by proteins acting in two major pathways, called homologous recombination and nonhomologous end-joining. the total number of genes influencing dsb repair efficiency is unknown. to identify new yeast genes affecting dsb repair, genes linked to gamma radiation resistance in previous genome-wide surveys were tested for their impact on repair of site-specific dsbs generated by in vivo expression of ecori endonuclease. several of the non-rad group genes have previously been linked to dna repair and over half of the genes affect nuclear processes. efficient mechanisms for rejoining of dna double-strand breaks are vital because misrepair of such lesions leads to mutation, aneuploidy and loss of cell viability. repair efficiency is also modulated by other processes such as sister chromatid cohesion, nucleosome remodeling and dna damage checkpoints. eight members of the rad group of dna repair genes and additional genes were found to be required for efficient repair of ecori-induced dsbs in screens utilizing both mata and matα deletion strain libraries. we have used a genetic screening approach to detect new genes required for efficient repair of dsbs in saccharomyces cerevisiae. many proteins encoded by the protective genes have previously been shown to associate physically with each other and with known dna repair proteins in high-throughput proteomics studies. ecorihomologous recombinationend-joiningdouble-strand breakbleomycinmmsradiationrad52gene ontology overlapping genes
 efficient mechanisms for rejoining of dna double-strand breaks are vital because misrepair of such lesions leads to mutation, aneuploidy and loss of cell viability. dsb repair is mediated by proteins acting in two major pathways, called homologous recombination and nonhomologous end-joining. repair efficiency is also modulated by other processes such as sister chromatid cohesion, nucleosome remodeling and dna damage checkpoints. the total number of genes influencing dsb repair efficiency is unknown.  to identify new yeast genes affecting dsb repair, genes linked to gamma radiation resistance in previous genome-wide surveys were tested for their impact on repair of site-specific dsbs generated by in vivo expression of ecori endonuclease. eight members of the rad group of dna repair genes and additional genes were found to be required for efficient repair of ecori-induced dsbs in screens utilizing both mata and matα deletion strain libraries. most mutants were also sensitive to the clastogenic chemicals mms and bleomycin. several of the non-rad group genes have previously been linked to dna repair and over half of the genes affect nuclear processes. many proteins encoded by the protective genes have previously been shown to associate physically with each other and with known dna repair proteins in high-throughput proteomics studies. a majority of the proteins share sequence similarity with human proteins, suggesting that they serve similar functions.  we have used a genetic screening approach to detect new genes required for efficient repair of dsbs in saccharomyces cerevisiae. the findings have spotlighted new genes that are critical for maintenance of genome integrity and are therefore of greatest concern for their potential impact when the corresponding gene orthologs and homologs are inactivated or polymorphic in human cells. ecorihomologous recombinationend-joiningdouble-strand breakbleomycinmmsradiationrad52gene ontology overlapping genes 