BACKGROUND
homologous recombination  is responsible for repairing damage affecting both dna strands and maintaining chromosome stability  <cit> .
in mammals, hr requires the rad <dig> family of proteins including rad <dig> and the rad <dig> paralogs   <cit> .
genetic studies have demonstrated that rad <dig> family members have non-redundant functions, as individual disruption of each gene confers increased sensitivity to dna damaging agents and a genome instability phenotype  <cit> .
in addition, the paralog proteins interact to form at least two stable complexes: a dimer consisting of rad51c-xrcc <dig> and a larger "bcdx2" complex consisting of rad51b, rad51c, rad51d, and xrcc <dig>  <cit> .
rad51d is unique among the rad <dig> family in that it is the only paralog currently known to support telomere maintenance in addition to the dna repair functions  <cit> .
alternative pre-mrna splicing is a mechanism responsible for proteome diversity and gene regulation in higher eukaryotes  <cit> .
splice variants of the rad51d gene have been reported previously in mouse and human tissues, as well as in cancer derived cell lines  <cit> .
similarly, rad51d alternative splice variants have also been identified in arabidopsis  <cit> .
for mus musculus, seven alternative transcripts were identified that are predicted to encode six distinct putative protein isoforms.
alternatively spliced translation products commonly display different or antagonistic biological functions compared to their full-length counterparts  <cit> .
therefore, changes in the pattern of alternative splicing of regulatory genes could have an impact on physiology and pathogenesis, particularly tumor development and progression  <cit> .
splice variants of dna repair genes potentially have the capability to regulate hr.
it has been demonstrated that two splice variants of rad <dig> increase the frequency of direct-repeat recombination from the same chromatid when expressed in either mammalian cells or yeast  <cit> .
moreover, mutations in the brca <dig> and brca <dig> genes, known to predispose carriers to breast and ovarian cancers, were found to disrupt exonic splicing enhancers and result in aberrant rna splicing  <cit> .
recently, a rad <dig> splice variant was uncovered that demonstrated homologous pairing activity similar to that of the full length rad <dig> protein  <cit> .
here, we report the mus musculus rad51d alternative transcripts encode predicted proteins capable of making specific interactions with rad51c and xrcc <dig> and the identification of two novel, ubiquitously expressed mus musculus rad51d alternative transcripts.
RESULTS
alternative transcripts of rad51d
multiple rad51d transcripts were first detected by northern blot analysis  <cit> , and seven splice variants were later identified by rt-pcr in both mouse and human brain tissues  <cit> .
the rad51d gene consists of  <dig> exons, and a summary of the current evidence for each alternative transcript for the human and mouse rad51d gene from the asd and eased databases is presented in table  <dig> <cit> .
the mus musculus rad51d alternative transcripts are summarized in figure 1a and for clarity are referred to as rad51dΔ  or rad51d+.
the highly conserved atp binding walker motifs a and b, present in all members of the rad <dig> family, are contained within exons  <dig> and  <dig> of rad51d respectively .
rad51d full length  includes both exons 7a and 7b in contrast to the rad51dΔ7b alternative transcript in which the final  <dig> base pairs of exon  <dig> are excluded.
previously, this 3' portion of exon  <dig> as well as the retained intron in rad51d+int <dig> were labeled as additional exons  <cit> .
internal deletions are also predicted in rad51dΔ <dig>  and rad51dΔ <dig> , while stretches of novel amino acid sequence and premature stop codons are predicted for the rad51dΔ <dig>  rad51dΔ <dig> and rad51d+int <dig> isoforms as a result of splicing induced frameshift mutations .
the splice variants rad51dΔ <dig> and rad51dΔ <dig> b are predicted to encode identical peptides.
rad51d isoforms interact with members of the bcdx <dig> complex
to investigate whether the predicted rad51d isoforms interact with binding partners of the full-length protein, each was examined for interaction with rad51c and xrcc <dig> by yeast two-hybrid analyses  <cit> .
yeast expressing the mouse rad51d-rad51c and rad51d-xrcc <dig> binding partners display growth on selective medium indicating strong protein interactions.
replica plating results suggest that rad51c interacts with rad51dΔ7b while xrcc <dig> interacts with rad51dΔ <dig>  rad51dΔ7b and rad51d+int <dig> .
the activating domain  fusion of rad51c suggested interaction with rad51dΔ <dig>  mouse rad51c-rad51c displayed positive growth when tested in both orientations of the gal <dig> fusion, which was not reported with the human rad51c protein  <cit> .
interaction between rad51c, xrcc <dig> and the alternatively spliced isoforms of rad51d was further assessed by measuring the activity of β-galactosidase.
colony lift assays were performed as a qualitative indicator of enzyme activity.
as illustrated in figure 2b, each of the matings that displayed growth on quadruple dropout medium were positive, with the exception of rad51c-rad51c.
β-galactosidase activity was weakest for the interactions of rad51dΔ <dig> and rad51dΔ <dig>  suggesting a weaker or transient interaction with rad51c and xrcc <dig>  to quantify the degree of binding between rad51d isoforms and rad51c/xrcc <dig>  β-galactosidase was measured using o-nitrophenyl-β-d-galactopyranoside as a substrate .
rad51dΔ7b displays a level of interaction with rad51c nearly identical to that of full-length rad51d.
in agreement with colony lift assays however, binding between rad51dΔ <dig> and rad51c was suggested in only one orientation.
rad51dΔ7b and rad51d+int <dig> associated with xrcc <dig> with an affinity similar to the full-length protein.
the rad51dΔ <dig>  rad51dΔ <dig> , and rad51dΔ <dig> isoforms display an ability to interact with xrcc <dig> , but variation in these interactions is observed depending upon the orientation of the gal <dig> fusion.
domain mapping of rad51d
the observation that rad51d isoforms may selectively interact with rad51c and xrcc <dig> allowed further domain mapping of rad <dig> paralog complexes.
miller et al.
reported that the amino-terminal domain of rad51d  interacts with xrcc <dig>  and the carboxy-terminal region  is sufficient for interaction with rad51c  <cit> .
binding of rad51dΔ <dig>  and rad51d+int <dig>  to xrcc <dig> support this observation .
however, the lack of association between rad51dΔ <dig> and rad51c suggests a more narrow region of the carboxy-terminal domain than previously reported, consisting of amino acids 234– <dig>  is required for the interaction between rad51d and rad51c.
interestingly, the yeast two-hybrid analyses displayed in figure  <dig> also suggest that residues 54– <dig> within the amino-terminal region determine whether rad51d interacts with xrcc <dig> .
to confirm these observations, the original rad51d deletion constructs used in the study by miller et al were tested against the rad51d isoforms.
if amino acids 54– <dig> are present, as with the rad51d  construct, the peptide interacts with xrcc <dig> .
when these residues are missing, as in the rad51dΔ <dig> isoform, there is an absence of association with xrcc <dig> and some interaction with rad51c.
this span of  <dig> amino acids  is nearly identical to the "linker region"  proposed from the structure modeling of human rad51d from the pyrococcus furiosus rad <dig> crystal structure  <cit> .
to further illustrate the position of the proposed region responsible for regulating these interactions, a protein homology model was constructed .
rad51d was matched to the rad <dig>  structure from pyrococcus furiosus  and was shown to have 24% identity and 46% similarity.
while the identity is somewhat low, the structural conservation of the rad <dig> family supports that meaningful models can be obtained.
after construction and refinement, the linker region is similar to what was proposed initially, but according to this model extends further into the alpha-helices of the n-terminal domain .
the overlapping region that potentially regulates rad51d interaction with xrcc <dig> and rad51c is located at the junction between the two domains previously demonstrated to interact with xrcc <dig> and rad51c respectively.
localization of rad51d isoforms
rad51d appears to be present throughout the cell although localization specific to telomeric regions in both meiotic and somatic cells has been demonstrated  <cit> .
to determine if the multiple isoforms of rad51d display similar localization, amino-terminal enhanced green fluorescent protein  tagged constructs were generated and transiently expressed in rad51d-deficient mouse embryonic fibroblasts  .
fluorescent microscopy reveals egfp-rad51d-fl is present in both the cytoplasm and nuclear compartments, similar to egfp vector control.
the rad51d alternatively spliced isoforms also display a mixed cytoplasmic and nuclear distribution within the cell.
in contrast, full length egfp-rad51c is predominantly nuclear in agreement with the presence of a non-canonical nuclear localization signal  in its carboxy-terminal region  <cit> .
full-length egfp-rad51d was tested for the ability to repair mitomycin c  induced dna damage in rad51d-deficient mefs.
the percentage resistance was nearly identical to that observed from cells transfected with rad51d-fl , suggesting the amino-terminal egfp fusion results in a biologically active protein.
additionally, no change in the localization of egfp-rad51d-fl was observed when expressed in wild-type mefs.
this confirms that failure to observe nuclear localization is not due to accumulated mutations in rad51d-deficient mefs .
complementation analysis of rad51d isoforms
expression of rad51d-fl was previously demonstrated to correct the dna interstrand crosslink repair deficiency of rad51d-deficientmefs  <cit> .
to determine whether rad51d alternative splice products retain any ability to restore cellular resistance to dna interstrand crosslinking damage caused by exposure to mmc, each was tested in a complementation assay  <cit> .
in the present study, approximately 45% of the population expressing full-length rad51d was capable of resisting mmc challenge .
however, none of the alternative rad51d isoforms restored resistance to dna interstrand cross-links when compared with vector control populations.
expression and identification of novel rad51d alternative transcripts
mouse rad51d alternative transcripts were originally identified in brain tissue  <cit> .
however, the isolation of corresponding expressed sequence tags in a variety of tissues and cell types suggest that rad51d isoforms are widely expressed .
to further investigate, rna isolated from c57bl/6j mouse tissues were used for rt-pcr expression analysis.
a series of overlapping pcrs were designed to detect all rad51d alternative transcripts .
rad51dΔ <dig> and rad51dΔ <dig> were consistently detected in most tissues examined and their identities verified by restriction enzyme and sequence analysis .
however, rad51dΔ <dig> was not detected in ovarian tissue.
additionally, rad51dΔ <dig> was not detected in mammary gland and testis, suggesting that these splice isoforms are differentially expressed.
because these same isoforms were identified in human adult and fetal brain cdna libraries  <cit> , initially named hstrad-d <dig> and hstrad-d <dig> respectively, expression analysis was performed in normal human breast tissue using primers that span from exon  <dig> to exon  <dig>  consistent with the results in mouse tissue, 21% of the detectable products corresponded to rad51dΔ <dig>  whereas rad51dΔ <dig> was not detectable above background levels .
as part of our efforts toward verifying the presence of exons  <dig> through  <dig> in rad51d splice variants  <cit> , 3'race was performed on total rna isolated from balb/c mouse liver tissue.
sequencing of cloned products revealed the existence of two additional alternatively spliced transcripts lacking sequence corresponding to one or both of the final rad51d exons .
both rad51dΔ <dig>  and rad51dΔ <dig> transcripts are derived from the use of alternative splice sites contained within downstream intronic sequence and predicted to result in c-terminal truncated rad51d isoforms.
notably for rad51dΔ <dig>  this alternative splice donor site is located  <dig> bp downstream, past the predicted rad51d polyadenylation site.
in addition, both rad51dΔ <dig>  and rad51dΔ <dig> predicted translation products contain a short stretch of novel sequence resulting from the ensuing frameshift.
using unique primers, both rad51dΔ <dig>  and rad51dΔ <dig> were demonstrated to be expressed in all tissues examined .
discussion
the studies described here are the first to explore functional properties of rad51d isoforms predicted to result from alternative splicing  <cit> .
each splice product was first tested for its ability to interact with rad51c and xrcc <dig>  proteins known to directly interact with rad51d and participate as part of the bcdx <dig> complex  <cit> .
bcdx <dig> is a complex that binds to dna nicks, which is one of the key steps during homologous recombination repair of dna crosslinks and branched structures, which are similar to holliday junctions  <cit> .
additionally, bcdx <dig> performs strand-annealing reactions  <cit> .
previous domain mapping of rad <dig> paralog interactions indicated that the amino-terminal portion of rad51d interacts with xrcc <dig> and the carboxy-terminal region interacts with rad51c  <cit> .
the rad51d+int <dig> isoform is predicted to contain residues 4– <dig> of the full-length protein and an additional  <dig> amino acids from the splice induced frameshift.
consistent with the miller model  <cit> , this isoform interacted strongly with xrcc <dig> and failed to interact with rad51c .
however, the rad51dΔ <dig> isoform containing the n-terminal domain, but not the linker region, did not interact with xrcc <dig> but did interact with rad51c, albeit dependent upon the orientation.
these results suggested that residues 60– <dig> within the rad51d predicted linker region governs interactions between the xrcc <dig> and rad51c paralogs.
therefore, within the cdx <dig> complex there may be three key interactions: 1) rad51c with both the n and c-terminal rad51d domains; 2) interactions of the rad51d linker region with xrcc; and 3) interactions between the n and c-terminal rad51d domains via a predicted salt bridge  <cit> .
electron microscopic analysis showed that rad51d-xrcc <dig> alone formed a multimeric ring structure in the absence of dna, similar to that of rad <dig>  but formed a filamentous structure unlike that of rad51/reca upon addition of ssdna  <cit> .
regulating these three types of interactions would likely influence the formation and function of this complex during hr repair.
the rad51dΔ7b isoform was the only alternative splice product to interact with both rad51c and xrcc <dig>  presumably, internal deletions resulting from rad51dΔ <dig>  and rad51dΔ <dig> affect integrity of the c-terminal domain, particularly the β-sheet  <cit> .
rad51dΔ7b is missing a small portion of exon  <dig>  resulting in an in-frame deletion of "gqqre" between residues 219– <dig>  because the deletion in rad51dΔ7b is in proximity to the adenine nucleotide binding sequence of walker motif b in rad51d  <cit> , it is possibly required for the hydrolysis of atp.
the atpase activity of rad51d is required for its role in the repair of dna interstrand crosslinks  <cit> .
residues adjacent to motif b have been found to be critical to atpase activity in walker box containing proteins including rad51d.
notably, conserved glutamate residues in atp-binding cassette proteins have been proposed to serve as a catalytic base in atp hydrolysis  <cit> .
thus, the observation that rad51dΔ7b did not complement sensitivity of rad51d-deficient cells from dna interstrand crosslinks caused by mitomycin c is consistent with a role for the "ggqre" sequence in atp hydrolysis.
the exclusion of an nls has been reported or implied from studies of numerous alternative splice products.
for example, predicted splice variants of both rad51c and rad51b are expected to lack their putative nls sequences  <cit> .
given the telomere binding of endogenous rad51d in human cells  <cit>  and the observation that each of the predicted isoforms of rad51d includes the "rkik" putative nls sequence at the amino terminus, nuclear localization of egfp-rad51d isoforms was expected.
in contrast, rad51d was expressed throughout the cell.
another published study, using egfp-rad51d, is consistent with this observation  <cit> .
it is conceivable that the presence of the egfp fusion in the amino-terminal region of rad51d inhibits access to the "rkik" sequence whereas the carboxy-terminal nls present in rad51c is unaffected.
alternatively, the over-expression of egfp-rad51d may saturate the cellular machinery responsible for import.
recently, a rad <dig> alternative splice isoform was identified which also lacks exons  <dig> and  <dig>  <cit> .
this hrad51-Δex <dig> had subtle differences in tissue expression, being primarily expressed in testis.
hrad51-Δex <dig> was also able to perform strand invasion activity similar to full-length and was localized to the nucleus, likely through an rkr motif introduced by the translational frameshift.
in either case, the likelihood remains that the "rkik" sequence in rad51d is not recognized as an nls, and transport of rad51d into the nucleus occurs as part of a protein complex  <cit> .
the previous protein modeling and interaction studies suggested linker regions were critical for interaction of the rad <dig> paralogs.
our results are consistent with this interpretation but also suggest this region may play a role in regulating these interactions.
a small region of rad51c was also identified to be critical for interaction with both rad51b and xrcc <dig>  <cit> .
it remains to be determined whether aberrant splicing of rad51d and related hr genes play a role during carcinogenesis.
however, two of the human alternative transcripts of rad51d have been identified in tumor derived cells   <cit> , including an est from a mammary tumor  that corresponds to the rad51dΔ <dig> isoform.
the association of alternative gene products in cancer raises the possibility that the alternative splicing mechanism is a potential target for gene therapy  <cit> .
molecular targeting of residues 54– <dig> in rad51d could disrupt interactions within the bcdx <dig> recombinosome and sensitize cells to dna interstrand crosslinks.
the pattern of protein-protein interactions between isoforms of rad51d and other rad <dig> paralogs described here suggests that rad51dΔ7b, rad51dΔ <dig>  and rad51d+int <dig> have potential to regulate homologous recombination repair by sequestering members of bcdx <dig> complex.
in addition, three of the alternatively spliced transcripts, rad51dΔ <dig>  rad51dΔ <dig>  and rad51d+int <dig>  harbor premature termination codons and are thus subject to degradation by nonsense-mediated mrna decay   <cit> .
more than one third of all mrnas derived from alternative splicing are estimated to contain similar nonsense codons  <cit> .
for that reason, it has been proposed that the pre-mrna splicing and nmd pathways are functionally coupled to provide an additional level of post-transcriptional regulation  <cit> .
therefore, cells could potentially alter gene expression by favoring the splicing of pre-mrna into an alternative transcript that is targeted to nmd.
it is also conceivable that hr may be directed on a tissue selective manner and that upregulation or downregulaton of specific isoforms provides a means of regulation.
this hypothesis is supported by the observation that the rad51d alternative transcripts display tissue specific expression patterns .
it is also intriguing that rad51d+int <dig> corresponds to one of the alternative splice isoforms predicted in humans  <cit> .
during the course of these studies, two new rad51d isoforms were isolated, rad51dΔ <dig> and rad51dΔ <dig> .
further studies are now necessary to explore the two newly discovered variants and determine whether splice isoforms may regulate hr.
finally, it remains to be determined whether these rad51d alternatively spliced products interact with additional known rad51d interacting proteins blm and sws <dig>  <cit>  or contribute to maintenance of telomere integrity.
CONCLUSIONS
here we report the mus musculus rad51d alternative transcripts encode predicted proteins capable of making specific interactions with rad51c and xrcc <dig>  expression studies revealed the rad51dΔ <dig> and rad51dΔ <dig> transcripts display tissue specific expression, being detected in each tissue except for mouse ovary or mammary gland and testis respectively.
additionally, we report the identification of two novel, ubiquitously expressed mus musculus rad51d alternative transcripts.
the predicted rad51d isoforms contain truncated c-terminal ends due to the retention of intron  <dig>  and the deletion of exon  <dig> .
the unusual number of alternative splice variants expressed from the rad51d gene compared with the other members of the rad <dig> family suggests the rad51d isoforms potentially regulate specific hr functions.

 homologous recombination is responsible for repairing damage affecting both dna strands and maintaining chromosome stability. in mammals, hr requires the rad family of proteins including rad and the rad paralogs. genetic studies have demonstrated that rad family members have non-redundant functions, as individual disruption of each gene confers increased sensitivity to dna damaging agents and a genome instability phenotype. in addition, the paralog proteins interact to form at least two stable complexes: a dimer consisting of rad51c-xrcc and a larger "bcdx2" complex consisting of rad51b, rad51c, rad51d, and xrcc. rad51d is unique among the rad family in that it is the only paralog currently known to support telomere maintenance in addition to the dna repair functions. alternative pre-mrna splicing is a mechanism responsible for proteome diversity and gene regulation in higher eukaryotes. splice variants of the rad51d gene have been reported previously in mouse and human tissues, as well as in cancer derived cell lines. similarly, rad51d alternative splice variants have also been identified in arabidopsis. for mus musculus, seven alternative transcripts were identified that are predicted to encode six distinct putative protein isoforms. alternatively spliced translation products commonly display different or antagonistic biological functions compared to their full-length counterparts. therefore, changes in the pattern of alternative splicing of regulatory genes could have an impact on physiology and pathogenesis, particularly tumor development and progression. splice variants of dna repair genes potentially have the capability to regulate hr. it has been demonstrated that two splice variants of rad increase the frequency of direct-repeat recombination from the same chromatid when expressed in either mammalian cells or yeast. moreover, mutations in the brca and brca genes, known to predispose carriers to breast and ovarian cancers, were found to disrupt exonic splicing enhancers and result in aberrant rna splicing. recently, a rad splice variant was uncovered that demonstrated homologous pairing activity similar to that of the full length rad protein. here, we report the mus musculus rad51d alternative transcripts encode predicted proteins capable of making specific interactions with rad51c and xrcc and the identification of two novel, ubiquitously expressed mus musculus rad51d alternative transcripts.  alternative transcripts of rad51d multiple rad51d transcripts were first detected by northern blot analysis, and seven splice variants were later identified by rt-pcr in both mouse and human brain tissues. the rad51d gene consists of exons, and a summary of the current evidence for each alternative transcript for the human and mouse rad51d gene from the asd and eased databases is presented in table. the mus musculus rad51d alternative transcripts are summarized in figure 1a and for clarity are referred to as rad51dΔ or rad51d+. the highly conserved atp binding walker motifs a and b, present in all members of the rad family, are contained within exons and of rad51d respectively. rad51d full length includes both exons 7a and 7b in contrast to the rad51dΔ7b alternative transcript in which the final base pairs of exon are excluded. previously, this 3' portion of exon as well as the retained intron in rad51d+int were labeled as additional exons. internal deletions are also predicted in rad51dΔ and rad51dΔ, while stretches of novel amino acid sequence and premature stop codons are predicted for the rad51dΔ rad51dΔ and rad51d+int isoforms as a result of splicing induced frameshift mutations. the splice variants rad51dΔ and rad51dΔ b are predicted to encode identical peptides. rad51d isoforms interact with members of the bcdx complex to investigate whether the predicted rad51d isoforms interact with binding partners of the full-length protein, each was examined for interaction with rad51c and xrcc by yeast two-hybrid analyses. yeast expressing the mouse rad51d-rad51c and rad51d-xrcc binding partners display growth on selective medium indicating strong protein interactions. replica plating results suggest that rad51c interacts with rad51dΔ7b while xrcc interacts with rad51dΔ rad51dΔ7b and rad51d+int. the activating domain fusion of rad51c suggested interaction with rad51dΔ mouse rad51c-rad51c displayed positive growth when tested in both orientations of the gal fusion, which was not reported with the human rad51c protein. interaction between rad51c, xrcc and the alternatively spliced isoforms of rad51d was further assessed by measuring the activity of β-galactosidase. colony lift assays were performed as a qualitative indicator of enzyme activity. as illustrated in figure 2b, each of the matings that displayed growth on quadruple dropout medium were positive, with the exception of rad51c-rad51c. β-galactosidase activity was weakest for the interactions of rad51dΔ and rad51dΔ suggesting a weaker or transient interaction with rad51c and xrcc to quantify the degree of binding between rad51d isoforms and rad51c/xrcc β-galactosidase was measured using o-nitrophenyl-β-d-galactopyranoside as a substrate. rad51dΔ7b displays a level of interaction with rad51c nearly identical to that of full-length rad51d. in agreement with colony lift assays however, binding between rad51dΔ and rad51c was suggested in only one orientation. rad51dΔ7b and rad51d+int associated with xrcc with an affinity similar to the full-length protein. the rad51dΔ rad51dΔ, and rad51dΔ isoforms display an ability to interact with xrcc, but variation in these interactions is observed depending upon the orientation of the gal fusion. domain mapping of rad51d the observation that rad51d isoforms may selectively interact with rad51c and xrcc allowed further domain mapping of rad paralog complexes. miller et al. reported that the amino-terminal domain of rad51d interacts with xrcc and the carboxy-terminal region is sufficient for interaction with rad51c. binding of rad51dΔ and rad51d+int to xrcc support this observation. however, the lack of association between rad51dΔ and rad51c suggests a more narrow region of the carboxy-terminal domain than previously reported, consisting of amino acids 234– is required for the interaction between rad51d and rad51c. interestingly, the yeast two-hybrid analyses displayed in figure also suggest that residues 54– within the amino-terminal region determine whether rad51d interacts with xrcc. to confirm these observations, the original rad51d deletion constructs used in the study by miller et al were tested against the rad51d isoforms. if amino acids 54– are present, as with the rad51d construct, the peptide interacts with xrcc. when these residues are missing, as in the rad51dΔ isoform, there is an absence of association with xrcc and some interaction with rad51c. this span of amino acids is nearly identical to the "linker region" proposed from the structure modeling of human rad51d from the pyrococcus furiosus rad crystal structure. to further illustrate the position of the proposed region responsible for regulating these interactions, a protein homology model was constructed. rad51d was matched to the rad structure from pyrococcus furiosus and was shown to have 24% identity and 46% similarity. while the identity is somewhat low, the structural conservation of the rad family supports that meaningful models can be obtained. after construction and refinement, the linker region is similar to what was proposed initially, but according to this model extends further into the alpha-helices of the n-terminal domain. the overlapping region that potentially regulates rad51d interaction with xrcc and rad51c is located at the junction between the two domains previously demonstrated to interact with xrcc and rad51c respectively. localization of rad51d isoforms rad51d appears to be present throughout the cell although localization specific to telomeric regions in both meiotic and somatic cells has been demonstrated. to determine if the multiple isoforms of rad51d display similar localization, amino-terminal enhanced green fluorescent protein tagged constructs were generated and transiently expressed in rad51d-deficient mouse embryonic fibroblasts. fluorescent microscopy reveals egfp-rad51d-fl is present in both the cytoplasm and nuclear compartments, similar to egfp vector control. the rad51d alternatively spliced isoforms also display a mixed cytoplasmic and nuclear distribution within the cell. in contrast, full length egfp-rad51c is predominantly nuclear in agreement with the presence of a non-canonical nuclear localization signal in its carboxy-terminal region. full-length egfp-rad51d was tested for the ability to repair mitomycin c induced dna damage in rad51d-deficient mefs. the percentage resistance was nearly identical to that observed from cells transfected with rad51d-fl, suggesting the amino-terminal egfp fusion results in a biologically active protein. additionally, no change in the localization of egfp-rad51d-fl was observed when expressed in wild-type mefs. this confirms that failure to observe nuclear localization is not due to accumulated mutations in rad51d-deficient mefs. complementation analysis of rad51d isoforms expression of rad51d-fl was previously demonstrated to correct the dna interstrand crosslink repair deficiency of rad51d-deficientmefs. to determine whether rad51d alternative splice products retain any ability to restore cellular resistance to dna interstrand crosslinking damage caused by exposure to mmc, each was tested in a complementation assay. in the present study, approximately 45% of the population expressing full-length rad51d was capable of resisting mmc challenge. however, none of the alternative rad51d isoforms restored resistance to dna interstrand cross-links when compared with vector control populations. expression and identification of novel rad51d alternative transcripts mouse rad51d alternative transcripts were originally identified in brain tissue. however, the isolation of corresponding expressed sequence tags in a variety of tissues and cell types suggest that rad51d isoforms are widely expressed. to further investigate, rna isolated from c57bl/6j mouse tissues were used for rt-pcr expression analysis. a series of overlapping pcrs were designed to detect all rad51d alternative transcripts. rad51dΔ and rad51dΔ were consistently detected in most tissues examined and their identities verified by restriction enzyme and sequence analysis. however, rad51dΔ was not detected in ovarian tissue. additionally, rad51dΔ was not detected in mammary gland and testis, suggesting that these splice isoforms are differentially expressed. because these same isoforms were identified in human adult and fetal brain cdna libraries, initially named hstrad-d and hstrad-d respectively, expression analysis was performed in normal human breast tissue using primers that span from exon to exon consistent with the results in mouse tissue, 21% of the detectable products corresponded to rad51dΔ whereas rad51dΔ was not detectable above background levels. as part of our efforts toward verifying the presence of exons through in rad51d splice variants, 3'race was performed on total rna isolated from balb/c mouse liver tissue. sequencing of cloned products revealed the existence of two additional alternatively spliced transcripts lacking sequence corresponding to one or both of the final rad51d exons. both rad51dΔ and rad51dΔ transcripts are derived from the use of alternative splice sites contained within downstream intronic sequence and predicted to result in c-terminal truncated rad51d isoforms. notably for rad51dΔ this alternative splice donor site is located bp downstream, past the predicted rad51d polyadenylation site. in addition, both rad51dΔ and rad51dΔ predicted translation products contain a short stretch of novel sequence resulting from the ensuing frameshift. using unique primers, both rad51dΔ and rad51dΔ were demonstrated to be expressed in all tissues examined. discussion the studies described here are the first to explore functional properties of rad51d isoforms predicted to result from alternative splicing. each splice product was first tested for its ability to interact with rad51c and xrcc proteins known to directly interact with rad51d and participate as part of the bcdx complex. bcdx is a complex that binds to dna nicks, which is one of the key steps during homologous recombination repair of dna crosslinks and branched structures, which are similar to holliday junctions. additionally, bcdx performs strand-annealing reactions. previous domain mapping of rad paralog interactions indicated that the amino-terminal portion of rad51d interacts with xrcc and the carboxy-terminal region interacts with rad51c. the rad51d+int isoform is predicted to contain residues 4– of the full-length protein and an additional amino acids from the splice induced frameshift. consistent with the miller model, this isoform interacted strongly with xrcc and failed to interact with rad51c. however, the rad51dΔ isoform containing the n-terminal domain, but not the linker region, did not interact with xrcc but did interact with rad51c, albeit dependent upon the orientation. these results suggested that residues 60– within the rad51d predicted linker region governs interactions between the xrcc and rad51c paralogs. therefore, within the cdx complex there may be three key interactions: 1) rad51c with both the n and c-terminal rad51d domains; 2) interactions of the rad51d linker region with xrcc; and 3) interactions between the n and c-terminal rad51d domains via a predicted salt bridge. electron microscopic analysis showed that rad51d-xrcc alone formed a multimeric ring structure in the absence of dna, similar to that of rad but formed a filamentous structure unlike that of rad51/reca upon addition of ssdna. regulating these three types of interactions would likely influence the formation and function of this complex during hr repair. the rad51dΔ7b isoform was the only alternative splice product to interact with both rad51c and xrcc presumably, internal deletions resulting from rad51dΔ and rad51dΔ affect integrity of the c-terminal domain, particularly the β-sheet. rad51dΔ7b is missing a small portion of exon resulting in an in-frame deletion of "gqqre" between residues 219– because the deletion in rad51dΔ7b is in proximity to the adenine nucleotide binding sequence of walker motif b in rad51d, it is possibly required for the hydrolysis of atp. the atpase activity of rad51d is required for its role in the repair of dna interstrand crosslinks. residues adjacent to motif b have been found to be critical to atpase activity in walker box containing proteins including rad51d. notably, conserved glutamate residues in atp-binding cassette proteins have been proposed to serve as a catalytic base in atp hydrolysis. thus, the observation that rad51dΔ7b did not complement sensitivity of rad51d-deficient cells from dna interstrand crosslinks caused by mitomycin c is consistent with a role for the "ggqre" sequence in atp hydrolysis. the exclusion of an nls has been reported or implied from studies of numerous alternative splice products. for example, predicted splice variants of both rad51c and rad51b are expected to lack their putative nls sequences. given the telomere binding of endogenous rad51d in human cells and the observation that each of the predicted isoforms of rad51d includes the "rkik" putative nls sequence at the amino terminus, nuclear localization of egfp-rad51d isoforms was expected. in contrast, rad51d was expressed throughout the cell. another published study, using egfp-rad51d, is consistent with this observation. it is conceivable that the presence of the egfp fusion in the amino-terminal region of rad51d inhibits access to the "rkik" sequence whereas the carboxy-terminal nls present in rad51c is unaffected. alternatively, the over-expression of egfp-rad51d may saturate the cellular machinery responsible for import. recently, a rad alternative splice isoform was identified which also lacks exons and. this hrad51-Δex had subtle differences in tissue expression, being primarily expressed in testis. hrad51-Δex was also able to perform strand invasion activity similar to full-length and was localized to the nucleus, likely through an rkr motif introduced by the translational frameshift. in either case, the likelihood remains that the "rkik" sequence in rad51d is not recognized as an nls, and transport of rad51d into the nucleus occurs as part of a protein complex. the previous protein modeling and interaction studies suggested linker regions were critical for interaction of the rad paralogs. our results are consistent with this interpretation but also suggest this region may play a role in regulating these interactions. a small region of rad51c was also identified to be critical for interaction with both rad51b and xrcc. it remains to be determined whether aberrant splicing of rad51d and related hr genes play a role during carcinogenesis. however, two of the human alternative transcripts of rad51d have been identified in tumor derived cells, including an est from a mammary tumor that corresponds to the rad51dΔ isoform. the association of alternative gene products in cancer raises the possibility that the alternative splicing mechanism is a potential target for gene therapy. molecular targeting of residues 54– in rad51d could disrupt interactions within the bcdx recombinosome and sensitize cells to dna interstrand crosslinks. the pattern of protein-protein interactions between isoforms of rad51d and other rad paralogs described here suggests that rad51dΔ7b, rad51dΔ and rad51d+int have potential to regulate homologous recombination repair by sequestering members of bcdx complex. in addition, three of the alternatively spliced transcripts, rad51dΔ rad51dΔ and rad51d+int harbor premature termination codons and are thus subject to degradation by nonsense-mediated mrna decay. more than one third of all mrnas derived from alternative splicing are estimated to contain similar nonsense codons. for that reason, it has been proposed that the pre-mrna splicing and nmd pathways are functionally coupled to provide an additional level of post-transcriptional regulation. therefore, cells could potentially alter gene expression by favoring the splicing of pre-mrna into an alternative transcript that is targeted to nmd. it is also conceivable that hr may be directed on a tissue selective manner and that upregulation or downregulaton of specific isoforms provides a means of regulation. this hypothesis is supported by the observation that the rad51d alternative transcripts display tissue specific expression patterns. it is also intriguing that rad51d+int corresponds to one of the alternative splice isoforms predicted in humans. during the course of these studies, two new rad51d isoforms were isolated, rad51dΔ and rad51dΔ. further studies are now necessary to explore the two newly discovered variants and determine whether splice isoforms may regulate hr. finally, it remains to be determined whether these rad51d alternatively spliced products interact with additional known rad51d interacting proteins blm and sws or contribute to maintenance of telomere integrity.  here we report the mus musculus rad51d alternative transcripts encode predicted proteins capable of making specific interactions with rad51c and xrcc expression studies revealed the rad51dΔ and rad51dΔ transcripts display tissue specific expression, being detected in each tissue except for mouse ovary or mammary gland and testis respectively. additionally, we report the identification of two novel, ubiquitously expressed mus musculus rad51d alternative transcripts. the predicted rad51d isoforms contain truncated c-terminal ends due to the retention of intron and the deletion of exon. the unusual number of alternative splice variants expressed from the rad51d gene compared with the other members of the rad family suggests the rad51d isoforms potentially regulate specific hr functions. 
however, none of the splice variants were capable of restoring resistance of rad51d-deficient cell lines to mitomycin c. rt-pcr expression analysis revealed that rad51dΔ and rad51dΔ transcripts display tissue specific expression patterns with rad51dΔ being detected in each tissue except ovary and rad51dΔ not detected in mammary gland and testis. the rad paralogs are essential for this process in vertebrates, and the rad51d paralog is unique in that it participates in both hr repair and telomere maintenance. rad51d splice variants have been reported in mouse and human tissues, supportive of a role for alternative splicing in hr regulation. these results suggest rad51d alternative splice variants potentially modulate mechanisms of hr by sequestering either rad51c or xrcc yeast-2-hybrid analysis was used to determine that the mus musculus rad51d splice variant product rad51dΔ7b was capable of interacting with both rad51c and xrcc and that rad51d+int interacted with xrcc in addition, the linker region of rad51d was identified as a region that potentially mediates binding with xrcc cellular localization, detected by egfp fusion proteins, demonstrated that each of the splice variant products tested was distributed throughout the cell similar to the full-length protein. the present study evaluated the interaction of the rad51d splice isoform products with rad51c and xrcc and their expression patterns.
 the homologous recombination pathway is vital for maintaining genomic integrity through the restoration of double-stranded breaks and interstrand crosslinks. the rad paralogs are essential for this process in vertebrates, and the rad51d paralog is unique in that it participates in both hr repair and telomere maintenance. rad51d is also known to directly interact with the rad51c and xrcc proteins. rad51d splice variants have been reported in mouse and human tissues, supportive of a role for alternative splicing in hr regulation. the present study evaluated the interaction of the rad51d splice isoform products with rad51c and xrcc and their expression patterns.  yeast-2-hybrid analysis was used to determine that the mus musculus rad51d splice variant product rad51dΔ7b was capable of interacting with both rad51c and xrcc and that rad51d+int interacted with xrcc in addition, the linker region of rad51d was identified as a region that potentially mediates binding with xrcc cellular localization, detected by egfp fusion proteins, demonstrated that each of the splice variant products tested was distributed throughout the cell similar to the full-length protein. however, none of the splice variants were capable of restoring resistance of rad51d-deficient cell lines to mitomycin c. rt-pcr expression analysis revealed that rad51dΔ and rad51dΔ transcripts display tissue specific expression patterns with rad51dΔ being detected in each tissue except ovary and rad51dΔ not detected in mammary gland and testis. these expression studies also led to the identification of two additional rad51d ubiquitously expressed transcripts, one deleted for both exon and and one deleted for only exon  these results suggest rad51d alternative splice variants potentially modulate mechanisms of hr by sequestering either rad51c or xrcc 