faithful dna replication is crucial for genomic maintenance . when replication is perturbed , cells activate stress response networks that connect the detection of replication - blocking lesions with dna damage tolerance ( ddt ) and repair pathways , chromatin modifications , cell - cycle control , and various other changes in cell physiology , often collectively referred to as the dna damage response ( ddr ) ( jackson and bartek , 2009 ) . failures in these processes are implicated in the etiology of many developmental and neurological disorders and are thought to drive genome instability characteristic of cancer ( odriscoll and jeggo , 2008 ) . genome duplication is carried out by the replisome machinery , initially assembled at replication origins ( gerbi and bielinsky , 2002 ) . notably , replication initiation critically depends on the loading and activity of the polymerase  ( pol)/primase complex . this is the fundamental initiator of dna replication in eukaryotic cells , as the replicative dna polymerases can only elongate an existing rna - dna primer produced by this complex . the primase produces short rna fragments ( about 712 nt long ) , which are subsequently subjected to limited extension by pol. these rna - dna primers are then extended by the replicative polymerases pol and pol ( aze et  al . , 2013 ) . pol/primase - mediated processes are not only relevant for origin - dependent replication initiation , but also for origin - independent initiation events , as is the case of lagging strand dna synthesis , and possibly the restart of stalled forks downstream the blocking lesion under conditions of genotoxic stress ( branzei and foiani , 2010 ; heller and marians , 2006 ) . the latter aspect is potentially crucial for efficient ddt and replication , especially in conditions in which fast replication is a requirement , such as at the early stages of development ( odriscoll and jeggo , 2008 ) . two distinct modes of ddt , error - prone and error - free ddt , operate in all eukaryotic organisms ( sale , 2012 ) . error - prone ddt is mediated by translesion synthesis ( tls ) polymerases and largely accounts for mutagenesis . error - free ddt uses a recombination - related mechanism known as template switching ( ts ) , in which one newly synthesized strand serves as replication template for the other blocked nascent strand ( branzei , 2011 ; giannattasio et  al . , 2014 ) . the choice between these ddt modes has profound consequences for genome stability , and to date , several factors have been implicated in ddt pathway choice : pcna post - translational modifications with mono - ubiquitylation , poly - ubiquitylation , and sumoylation ( branzei et  al . , 2008 ; 2005 ) ; genome architectural transitions coupled with early stages of replication ( gonzalez - huici et  al . , 2014 ) ; and cell - cycle - specific changes in the abundance or regulation of key ddt factors ( karras et  al . , 2013 ; waters and walker , 2006 ) . together with pol/primase , a number of structural proteins that tether the replicative minichromosome maintenance ( mcm ) helicase to the replicative polymerases are loaded at replication origins ( aze et  al . , 2013 ) . ctf4 ( and-1 in mammalian cells ) functions as such a replisome architectural factor , bridging the mcm helicase and two molecules of pol/primase ( gambus et  al . , 2009 ; simon et  al . , it is of note , however , that while pol and primase are essential for cellular proliferation , ctf4 is not . this indicates that even if uncoupled from the replicative helicase , pol/primase supports dna synthesis . besides its roles to maintain normal replisome architecture , ctf4/and-1 is also required for sister chromatid cohesion ( hanna et  al . , 2001 ; yoshizawa - sugata and masai , 2009 ) . increasing number of reports indicate  replication stress  at the basis of chromosomal instability , and as an important underlying factor of developmental anomalies ( halazonetis et  al . , 2008 ; however , the nature of the early chromosome lesions arising following such replication perturbations is largely unknown . moreover , the connections between these replication dysfunctions and the observed chromatin structural alterations similarly triggered by mutations in cohesion factors remain elusive . here we used budding yeast saccharomyces cerevisiae cellular models of specific replication stress and sister chromatid cohesion defects to investigate a possible crosstalk between recombination - mediated ddt and chromatin structure / cohesion . our results indicate that both replicative helicase - coupled re - priming and sister chromatid cohesion are important to facilitate error - free ddt by ts , but they do so via different mechanisms . the results shed light on how highly conserved replication - associated pathways crosstalk to each other and contribute to normal replication fork and chromatin structure , providing mechanistic insights into the molecular basis of human disorders caused by replication dysfunctions . error - free ddt by ts can be molecularly monitored by examining the formation of x - shaped structures composed of sister chromatid junctions ( scjs ) in the proximity of replication forks using 2d gel electrophoresis ( branzei et  al . , 2008 ) . in this assay , yeast cells are released synchronously and allowed to replicate in media containing the alkylating reagent methyl methanesulfonate ( mms ) . the pattern of replication intermediates at genomic locations of interest is analyzed at different time points during dna replication . previous results showed that ts intermediates form during replication of damaged templates and accumulate when the sgs1-top3-rmi1 complex is defective because of compromised resolution ( branzei et  al . , 2008 ; liberi et  al . , 2005 ; giannattasio et  al . , replication of damage templates leads to a progressive accumulation of scjs in sgs1 mutant cells ( liberi et  al . , 2005 ) , as well as in sgs1 hypomorphic mutants disrupted only in the helicase domain ( onoda et  al . deletion of ctf4 causes a g2/m delay associated with slower proliferation ( kouprina et  al . , 1992 ) , but it does not prominently impact on s phase progression when cells are replicating in the presence of mms ( figure  s1a ) . notably , we found that the ctf4 mutation reduced the amount of x - shaped scjs in both sgs1 and wild - type ( wt ) background ( figures 1a and s1b ) , suggesting a supportive role for ctf4 in ts . cells defective in error - free ddt are characterized by a more  prominent dependency on tls , showing higher levels of spontaneous mutagenesis and increased damage sensitivity  following inactivation of error - prone ddt components ( cejka et  al . , 2001 ) . we found that ctf4 cells have statistically significant increased spontaneous mutagenesis rates ( figure  1b ) . this was largely dependent on rev3 ( figure  1b)the catalytic subunit of the tls polymerase pol , accounting for most of the mutagenic events in budding yeast . furthermore , ctf4 and rev3 showed strong additive effects for mms sensitivity ( figure  s1c ) , supporting the view that tls - mediated ddt acts as a compensatory mechanism in ctf4 cells . to further characterize the role of ctf4 in ddt , we examined its genetic interactions with classical error - free ddt pathways governed by rad6-rad18 post - replicative repair and rad51 recombinational repair branches . ctf4 mutants showed additive damage sensitivity when combined with rad18 or rad51 ( figure  s1d ) . since the rad6-rad18 pathway controls both ts- and tls - mediated ddt , we also examined the genetic interaction of ctf4 with the pcna polyubiquitylation pathway ( mediated by rad5 , mms2 , and ubc13 ) that specifically affects ts ( pfander et  al . , 2005 ) . however , also in this case , we found an additive effect on damage survival ( figure  s1e ) . next , to address the possibility of an effect of ctf4 on ddt by influencing the post - translational modifications of pcna , we analyzed the  pattern of sumo- and ubiquitin / polyubiquitin - conjugated pcna species in ctf4 cells . we detected no major changes in pcna modifications both in spontaneous and mms - treated conditions ( figure  1c ) . we conclude that ctf4 facilitates error - free ddt in parallel with canonical post - replicative repair pathways regulated by pcna modifications with sumo and ubiquitin . previous work assigned two main functions to ctf4 : an architectural role in the context of the replisome  achieved by bridging the pol/primase complex to the mcm replicative helicase ( gambus et  al . , 2009 ; simon et  al . , 2014 ; tanaka et  al . , 2009)and an additional role in sister chromatid cohesion ( hanna et  al . , a ctf4 deletion mutant shows a mild increase in premature sister chromatid separation in g2/m , accompanied by sporadic chromosome loss and aneuploidy without obvious defects in completing the bulk of dna replication ( hanna et  al . , 2001 ; it is therefore possible that the ts defect of ctf4 mutants ( figure  1a ) is an indirect consequence of defective sister chromatid cohesion or , alternatively , a direct result of mcm - uncoupled pol/primase compromised activity . the first hypothesis leads to the proposition that mutants defective in cohesion , but proficient for pol/primase function , would also be defective in formation of ts intermediates composed of scjs . to test this , we analyzed the consequences for ts of impairing sister chromatid cohesion using genetic conditions that result in loosening or opening of the cohesin ring . scc1 is an essential subunit of the cohesin complex that is cleaved at anaphase ( uhlmann et  al . , 1999 ) . using the temperature - sensitive allele , scc1 - 73 , we found that impairment of cohesin function during a single round of replication also caused a defect in ts ( figures 2a ) . similar results were obtained using a gal promoter - driven conditional scc1 allele , gal - scc1 , the expression of which is induced by galactose and repressed by glucose ( data not shown ) . next , we asked if pol/primase mutants are different or similar to ctf4 and cohesin mutants with respect to ts . we investigated the contribution of two alleles , pri1-m4 , affecting the pri1 subunit of primase ( marini et  al . , 1997 ) , and pol1 - 1 , affecting the catalytic subunit pol1 of pol ( lucchini et  al . , 1988 ) . to mildly interfere with the function of pol/primase without affecting the ability of cells to traverse s phase , cells were grown at the permissive temperature and synchronously released in media containing mms at semi - permissive temperatures that allow completion of dna replication with normal kinetics ( data not shown ; see below ) . both pri1-m4 and pol1 - 1 mutations caused a reduction in the x molecules accumulating in sgs1 ( figures 2b and s2a ) . this phenotype is suggestive of a positive role of pol/primase in error - free ddt by ts , a conclusion also supported by the increased mutagenesis in pol and pri1 mutants previously reported ( longhese et  al . , 1993 ; considering the phenotypic similarity between ctf4 and both cohesin and pol/primase mutants with regard to ts , we asked if ctf4 contribution to cohesion might take place in the context of the pol/primase complex . to this purpose , we examined the percentage of premature sister chromatid separation , a widely used cohesion readout , in pri1-m4 cells . compared to wt , pri1-m4 cells exhibited a significant increase in premature sister chromatid separation , at a level similar with the one caused by mutations in the non - essential cohesion factors ctf4 , chl1 , and ctf18 included in the analysis ( figure  2c ) .  previously , based on genetic interactions , non - essential cohesion factors were divided in two genetic pathways , having as prominent members ctf4 and chl1 or ctf18 and ctf8 , respectively ( xu et  al . , while combination of mutations in the same cohesion pathway generally does not exacerbate the observed cohesion defects associated with single mutations  and does not lead to synthetic fitness defects , the opposite  is  true for combinations of mutations belonging to separate pathways . to further test if the cohesion defects observed with pri1-m4 ( figure  2c ) are manifested in the context of the pol/primase / ctf4 complex , we combined pri1-m4 with deletion mutations in genes affecting the two non - essential cohesion pathways . supporting the above view that pri1-m4 cohesion defects reflect a role for pol/primase / ctf4 in cohesion , pri1-m4 manifested synthetic fitness defects with ctf18 and ctf8 , but not with ctf4 or chl1 ( figure  s2b ; data not shown ) . altogether , these data demonstrate that interfering with the functionality of the pol/primase / ctf4 and cohesin complexes results in similar defects both in cohesion and error - free ddt by ts , leading to the question of whether cohesion and ts recombination are linked by a causal relationship . sister chromatid cohesion defects reflect impairments in maintaining accurate physical proximity between the newly replicated chromatids . while it is reasonable to assume that by influencing the accessibility of the sister chromatid donor sequence cohesin facilitates sister chromatid recombination ( covo et  al . , 2010 ; tittel - elmer et  al . , 2012 ) , to what extent the physical proximity of chromatids impacts on scj formation during the non - canonical recombination mechanism of ts is unknown . to address this , we used a previously described system that artificially re - establishes cohesion in a locus - specific manner . the system relies on the ability of the lactose inhibitor ( laci ) to bind the lactose operator sequence ( laco ) . in the wt form ( tetramer ; figure  3a , right ) , laci is able to form tetramers that bind two different laco sequences present on each chromatid , while a truncated version of the laci protein ( dimer ; figure  3a , left ) is only able to form a dimer and to bind the laco sequences on a single chromatid . therefore , after dna replication , only the tetramer version of laci , and not the dimer , can bind the identical laco sequences placed on both sister chromatids , restoring sister chromatid cohesion locally in cohesion - defective mutants ( straight et  al . , 1996 ) . by using the above assay , we examined whether artificial tethering of the sister chromatids was able to locally rescue the ts defects associated with cohesin mutants . sgs1 and sgs1 scc1 - 73 cells carrying either the dimer or the tetramer laco / laci system were synchronized at the permissive temperature , and after activation of laci expression , cells were released in media containing mms at the non - permissive temperature for scc1 - 73 to induce ts defects associated with decreased cohesin function ( see figure  2a ) . next , we examined by 2d gel the pattern of ts intermediates arising at locations proximal to the laco array ( figure  2a ) . we found that , unlike the sgs1 scc1 - 73 mutant carrying the dimer version of laci , which showed reduced accumulation of ts intermediates ( figure  3b , upper panel ) , the strain with the tetramer version of laci rescued x molecule formation ( figure  3b , bottom panel ) . to verify the specificity of the tethering system , we digested and hybridized the same genomic dna to probe for replication intermediates forming in the proximity of the early origin of replication ars1 , located on chromosome iv , which should not be affected by the laci variants binding to laco cassettes on chromosome iii . as expected , the ts defect associated with scc1 - 73 at ars1 was not alleviated ( figure  s3 ) . because artificial tethering of the chromatids is able to bypass the absence of a functional cohesin ring in regard to scj formation , these experiments demonstrate that the role exerted by the cohesin complex in ts is structural . to now assess whether , as in scc1 - 73 , the ctf4 defect in ts is also due to increased physical distance between the sister chromatids , we used the same experimental approach , but in the sgs1 ctf4 background . interestingly , artificial cohesion did not restore the formation of x molecules in the sgs1 ctf4 strain ( figure  4a ) , although the laco / laci system efficiently reduced the local cohesion defects on chromosome iii associated with ctf4 ( figure  4b ) . the function of ctf4 in ts  revealed by this assay is thus different from the structural one of cohesin ( figure  3b ) . of note , the defect of ctf4 cells resembled the one caused by inactivation of the rad51 recombinase ( figure  s4a ) . these results indicate that ctf4 affects key  aspects of the ts reaction that can not be substituted by simply restoring the physical proximity of the sister chromatids . taken together , the data suggest that the repair / ts defect of ctf4 mutants is primarily caused by faulty mcm - uncoupled pol/primase activity rather than by defective sister chromatid cohesion . based on the above results , we conclude that , at least in regard to ddt , the roles of cohesin and pol/primase / ctf4 are manifested via two fundamentally distinct mechanisms . supporting this view , deletion of ctf4 resulted in synthetic lethality when combined with scc1 - 73 even at permissive temperatures ( figure  s4b ) ( mclellan et  al . , 2012 ) . in addition , using a combination of temperature - sensitive alleles of pol/primase mutants and cohesin , we obtained viable pri1-m4 scc1 - 73 cells that displayed , however , increased mms sensitivity compared to the single mutants ( figure  s4c ) . this result highlights once again a differential contribution of cohesin and the pol/primase / ctf4 complex toward ddt . a diverse molecular purpose of pol/primase and cohesin during canonical recombination can also be deduced from their contribution to the donor choice : in cohesin mutants , the inter - sister recombination events decrease in favor of events involving the homologous chromosome ( covo et  al . , 2010 ; tittel - elmer et  al . , 2012 ) , whereas ctf4 causes a decrease in both sister- and inter - homologous recombination in conditions of dna damage ( ogiwara et  al . , these results reveal that , despite cohesin and ctf4 mutants showing similar phenotypes in cohesion and ddt , cohesin aids ts by maintaining the proximity of the recombination donor , while the pol/primase / ctf4 complex must affect some fundamental activity or step during recombination - mediated damage bypass . to identify the ts - sensitive step that is defective in pol/primase / ctf4 complex mutants , when re - priming is either affected or uncoupled from the mcm helicase , we combined ctf4 and pri1-m4 with a series of mutations affecting distinct hr - associated processes . we found that ctf4 shows synthetic sickness / lethality with rad52 ( kouprina et  al . , 1992 ) , but not with rad51 ( figure  5a ) or other rad51 mediators , such as rad55 , that assist rad51-mediated strand invasion ( data not shown ) . we also observed a similar pattern of genetic interactions for pri1-m4 ( figure  s5a ) . although rad51 and rad52 act together in most recombination events , a significant amount of hr events are detectable in rad51 mutants that are dependent on rad52 ( krogh and symington , 2004 ) . in saccharomyces cerevisiae , the major rad51-independent activity of rad52 is represented by strand annealing , which also involves rad59 and rpa ( krogh and symington , 2004 ) . notably , ctf4 also showed synthetic fitness defects when combined with rad59 ( figure  5b ) and the s.  cerevisiae rpa largest subunit rfa1 allele , rfa1-t11 ( figures 5c ; see below ) . however , no synthetic interactions were observed with rad1 ( figure  s5b ) , defective in the process of double - strand break ( dsb ) repair by single - strand annealing ( krogh and symington , 2004 ) . the single - stranded dna - binding complex rpa initially covers ssdna in order to prevent secondary structure formation and to mediate the subsequent binding of rad52 ( krogh and symington , 2004 ) . binding of rad52 to stretches of rpa - covered ssdna may promote annealing of such sequences , potentially leading to rearrangements , such as deletions and duplications , especially in the presence of repeat sequences ( branzei , 2011 ) . supporting this view , we found a strong increase , of a 1,000-fold magnitude , in spontaneous deletion rates of genomic elements flanked by repeat sequences , in both ctf4 and pri1-m4 mutants with respect to wt ( figure  5d ) . these deletion events were not dependent on rad51 ; rather , the rad51 mutation itself caused an increase in such deletion events in both wt and ctf4/pri1-m4 backgrounds ( figure  5d ) . to directly test the genetic evidence of an altered amount of ssdna in ctf4 and pri1-m4 mutants , we performed electron microscopy ( em ) analysis on the replication intermediates extracted from wt , ctf4 , and pri1-m4 cells replicating under damaging conditions . pri1-m4 showed a strong increase in the length of the ssdna stretches exposed at the replication fork ( figure  6a ) . in addition to this , we found that although not different for the average length , both ctf4 and pri1-m4 mutants showed a 2-fold increase in the number of internal gaps detected behind replication forks ( figure  6b ) . holliday junction - like structures in which the two newly synthesized strands are paired , in both ctf4 and pri1-m4 ( figure  6c ) . it is of note that such reversed fork structures were absent in wt cells , suggesting that reversed forks are not predominantly induced following genotoxic stress in saccharomyces cerevisiae . this is in line with previous reports on total replication intermediates formed during replication in the presence of uv- or mms - induced damage ( lopes et  al . , 2006 ) , and with the structural analysis of x - shaped structures forming at a specific chromosome locus during replication of damaged templates in wt and sgs1 budding yeast cells ( giannattasio et  al . , 2014 ) altogether , the results demonstrate a profound alteration in the metabolism of ssdna in pol/primase / ctf4 complex mutants . this likely leads to the subsequent formation of unscheduled reversed forks and recombination structures , having negative effects on both chromatin structure and ddr . this process generates endogenous damage and replication stress , although the molecular nature of the latter remains poorly understood . while various studies outline that intimate links must exist between dna replication and ddt / ddr mechanisms ( branzei and foiani , 2010 ; jackson and bartek , 2009 ) , how relatively mild replication - challenging conditions interfere with other chromosome metabolism processes associated with dna replication remains largely elusive . here we set out to examine how specific replication pathways may crosstalk to each other and affect ddt . remarkably , we found that mild impairment in replication - associated molecular pathways related to re - priming , replisome architecture , and cohesion invariably negatively impinged on template switching ( figures 1a and 2b ) . the defects in error - free ddt were coincident with an increase in mutagenic ddt and other types of genomic rearrangements ( figures 1b and 5d ) , as well as with altered replication fork structures ( figure  6 ) . these results provide in  vivo support for the primer - driven post - replicative ddt as the most prominent pathway of ddt , at least in budding yeast ( becker et  al . , 2014 ; branzei , 2011 ; heller and marians , 2006 ; lehmann and fuchs , 2006 ) . moreover , as deletion of ctf4 that does not directly impact on primase activity caused a similar phenotype to the one of primase mutants , the present work carries the implication that this re - priming function provides genome stability when carried out in the context of mcm - coupled pol/primase protein complex ( figure  7 ) . on the contrary , when re - priming is deregulated , problems are bound to occur . limited re - priming can lead to fork uncoupling and long stretches of ssdna at the fork ( figure  6a ) ; these ssdna discontinuities can engage in error - prone annealing events ( figure  5d ) and cause altered replication fork architecture ( figures 6c and  7 ) . deregulated re - priming events can lead to a larger number of internal gaps ( figure  6b ) , some of which may be more easily filled - in via tls - mediated mechanisms ( figure  1b ) or also engaged by error - prone strand - annealing activities ( figures 5d and 7 ) . notably , under conditions of limited or mcm - uncoupled re - priming , we observe an unusual high frequency of fork - reversal events associated with long ssdna stretches at the fork . the ssdna regions may be due to prolonged replication fork uncoupling or exonucleolytic processing of the reversed forks ( figure  7 ) . these observations indicate that  at least in saccharomyces cerevisiae  fork reversal is disfavored or extremely transient when re - priming is working efficiently . the fork reversal phenotype we reported here for ctf4 and pri1-m4 mutants is induced by dna damage . while in primase mutants about 12% of the forks are present in reversed conformation during replication in the presence of mms ( figure  6c ) , following fork stalling induced by hu treatment , we found only 1% reversed forks in primase mutants and no reversed forks in wt and ctf4 mutants ( data not shown ) . this is in contrast with the phenotype of rad53 replication checkpoint mutants in which about 10% of replication forks are in a reversed fork conformation after hu treatment ( cotta - ramusino et  al . , 2005 ; sogo et  al . , 2002 ) . accumulation of reversed forks upon checkpoint defects likely reflects local accumulation of torsional stress ( bermejo et  al . , 2011 ) and not ddt attempts , as only about 1% reversed forks are observed in checkpoint defective cells upon mms treatment ( lopes et  al . , 2006 ) . thus , replication fork reversal is differentially modulated upon challenges to replisome stability or during replication - associated ddt . under specific types of genotoxic stress ( ray chaudhuri et  al . , 2012 ) , or upon impairment of kinetically favored ddt mechanisms ( e.g. , re - priming ) , transient fork reversal may become a crucial strategy to promote fork stabilization or to mediate alternate modes of damage - bypass in both s.  cerevisiae and higher eukaryotic cells ( neelsen and lopes , 2015 ) . while fork reversal may initially function as a fork stabilization mechanism both at stalled and damaged replication forks ( atkinson and mcglynn , 2009 ; neelsen and lopes , 2015 ) , it is of note that  unless promptly restarted  reversed forks can be processed by multiple nucleases ( cotta - ramusino et  al . , 2005 ; doksani et  al . , 2009 ; neelsen et  al . , 2013 ; szakal and branzei , 2013 ) , thus being a potential source of deleterious ssdna discontinuities and dsbs that may trigger , in the long run , genome instability ( figure  7 ) . indeed , reduced amounts of pol in saccharomyces cerevisiae induce recombination - associated chromosome deletions and duplications ( song et  al . , 2014 ) . we propose that altered fork structures may be engaged in break - induced replication ( bir ) type of mechanisms ( figure  7 ) , which may function as alternate and likely aberrant modes of ddt , prone to deletions as well as genomic duplications ( costantino et  al . , 2014 ) . notably , in support of the bir notion above , we found that ctf4 and pri1-m4 mutants display synthetic lethality with deletions of rad52 and rad59 , generally required for bir , but not with rad51 , which is often dispensable for bir ( anand et  al . , 2013 ) . as many replication factors , including ctf4 , also  facilitate the dna synthesis step of bir ( lydeard et  al . , 2010 ) , it is likely that the bir events induced under such replication stress conditions might be more prone to errors , leading to rearrangements . indeed , inactivating mutations in error - free ddt factors as well as in pol/primase cause an increase in genomic duplications and deletions ( figure  5d ) ( putnam et  al . , 2010 ) . altogether , these results provide a molecular rationale for why cellular conditions characterized by replication stress are coincidently defective in error - free ddt processes and prone to deleterious bir or other annealing events ( figures 1 and 5 ) . these recombination pathways are bound to involve similarly defective replication steps , thus setting the stage for vicious circles in which more replication stress is being created . mild replication dysfunctions of the type we uncovered here in ctf4 and pri1-m4 mutants are likely the culprits not only for genome instability , but also for chromosomal structural and cohesion alterations . indeed , we found that differently from impairments in cohesin , the cohesion and ts defects of pol/primase / ctf4 are in a non - causal relationship . on one hand , improvement of sister chromatid proximity does not rescue the ts defects of ctf4 mutants ( figure  4 ) . on the other hand , inability to perform ts is unlikely at the basis of the observed cohesion defects , as mutations in canonical ts factors do not result in similar cohesion defects ( warren et  al . , 2004 ) . we propose that the two phenotypes , defective ddt and cohesion impairment , arise coincidentally , but likely independently , from the same replication condition . in the case of pol/primase / ctf4 mutants , the underlying replication dysfunction relates to deregulated re - priming , and abnormal distribution of ssdna discontinuities . in addition , it is possible that increased replication pausing and altered fork structure in pol/primase / ctf4 complex mutants affects the topology of the replicating dna in manners that negatively impinge on cohesion ( liu et  al . , 2010 ) . the coincident cohesion and ddt defects associated with mutations in the pol/primase / ctf4 complex suggest an intimate relationship between these two biological processes during replication . this hypothesis may also explain that hypomorphic mutations in other replication factors , affecting replication initiation or primer - processing , lead to cohesion defects and reduced tolerance to genotoxic stress ( kerzendorfer et  al . remarkably , oncogene - induced replication stress , as well as ddr and cohesion dysfunctions associated with neurological and developmental defects , resemble in many respects the replication impairments studied here ( halazonetis et  al . , 2008 ; neelsen et  al . , 2013 ; odriscoll and jeggo , 2008 ; van der lelij et  al . , 2010 ) . we propose that aberrant ddt associated with compromised error - free ts and fork rearrangements is a potential underlying common source of lesions in a number of replication disorders . the yeast strains used in this study were mostly derivatives of w303 and detailed genotypes are indicated in table s1 . information about growing media , cell cycle arrest , release , and analysis are described in supplemental experimental procedures . forward mutation and spontaneous intra - chromosomal deletion rates were calculated as described in the supplemental experimental procedures . purification of dna intermediates and 2d gel analysis were carried out as previously described ( vanoli et  al . , 2010 ) . each experiment shown was performed independently at least twice with qualitatively identical results . genome preparation and signal quantification methods
summarychromosomal replication is entwined with dna damage tolerance ( ddt ) and chromatin structure establishment via elusive mechanisms . 
 here we examined how specific replication conditions affecting replisome architecture and repriming impact on ddt . 
 we show that saccharomyces cerevisiae pol/primase / ctf4 mutants , proficient in bulk dna replication , are defective in recombination - mediated damage - bypass by template switching ( ts ) and have reduced sister chromatid cohesion . 
 the decrease in error - free ddt is accompanied by increased usage of mutagenic ddt , fork reversal , and higher rates of genome rearrangements mediated by faulty  strand annealing . 
 notably , the ddt defects of pol/primase / ctf4 mutants are not the consequence of increased sister chromatid distance , but are instead caused by  altered single - stranded dna metabolism and abnormal replication fork topology . 
 we propose that error - free ts is driven by timely replicative helicase - coupled re - priming . 
 defects in this event impact on replication fork architecture and sister chromatid proximity , and represent a frequent source of chromosome lesions upon replication dysfunctions .