TI - DISCUSSION . AB - In this study , we analyzed the multiple start codons present in the S.cerevisiae RAD52 ORF by generating a series of mutations within its N-terminus . We conclude that translation of RAD52 is initiated from three start codons and each of the resulting protein species are competent in DNA repair and homologous recombination . According to the 'scanning' model ( 17-20 ) , translation should initiate preferentially when the ribosome encounters the first AUG start site . In the case of S.cerevisiae RAD52 , the mRNA transcript begins after the second ATG codon ( 15 ) ; therefore , the third ATG codon corresponds to the first translational start site that can be encountered . However , owing to the suboptimal context around the third start site ( a T at the -3 position ) , ribosome slippage at this site likely results in protein expressed from the fourth ATG triplet , which has an optimal A at the -3 position . Alternatively , initiation at the fourth or fifth AUG could be due to shunting or internal ribosome entry [for a review see ( 51 ) ] . Moreover , the small amount of Rad52 protein synthesized in the FS4-5 strain suggests that leaky scanning by the ribosomes occurs to bypass start codons three and four and initiate from the fifth ( Figure 4 , lane 8 ) . By eliminating translation initiation from codons three and four ( E24Stop M34A M38A ) , wild-type levels and wild-type activity are seen for Rad52 protein made exclusively from the fifth start codon ( Figure 3D ; Figure 4 , lane 10 and Table 3 ) . The increased gamma-ray resistance of a strain with protein translated only from the fifth start codon (E24Stop M34A M38A) compared to the FS4-5 strain indicates that efficient repair of gamma-ray-induced lesions requires wild-type Rad52 protein levels . This notion is further supported by the observations that a 3 - to 4-fold reduction in wild-type Rad52 levels in the rad52-Y66oSUP4-o strain results in a 4-fold decrease in spontaneous recombination as well as a 2 - to 3-fold increase in gamma-ray sensitivity ( Table 3 and data not shown ) . It was proposed previously that Rad52 protein levels affect heteroallelic recombination more than the repair of gamma-ray-induced lesions ( 36 ) . However , that conclusion was based on a class of Rad52 mutants (Class D) that contained both a missense mutation as well as decreased protein levels . In light of the results described above , it is likely that protein levels equally affect recombination and repair . Since the first and second ATG codons are not part of the RAD52 -encoded mRNA , then the third ATG codon in the S.cerevisiae RAD52 ORF is the first potential AUG codon in the mRNA . However , for simplicity and consistency with previous work involving various rad52 mutant strains ( 36,48-50 ) , throughout this report and in the future , we will continue to number amino acid positions from the first ATG codon based on the RAD52 ORF predicted by the DNA sequence rather than the first AUG of the RAD52 -encoded mRNA . Multiple Rad52 bands are observed where Rad52 can only be translated from either start site three or five ( Figure 4 , lanes 8-10 ) . Although previous results indicated that protein mobilities of the multiple Rad52 species were not altered after treatment with calf intestinal alkaline phosphatase ( 45 ) ; in this study , we demonstrate that Rad52 protein is indeed PHOSphorylated and this modification occurs in a cell cycle -independent and cell cycle -dependent manner at serine and/or threonine residues . For example , wild-type cells that are synchronized and released from a G1 arrest exhibit multiple Rad52 bands that after dePHOSphorylation collapse to a doublet band corresponding to translational products starting from codon three and from codons four and five , which co-migrate ( Figure 5C ) . In addition , both the cell cycle -independent and cell cycle -dependent phosphorylaTION events require the C terminal domain of Rad52 (Figure 5D) , suggesting that Rad52 is PHOSphorylated at the C terminus . Alternatively , Rad52 may be PHOSphorylated within the N-terminal domain but its modification requires the presence of the C-terminus . One of the known functions of the C-terminal domain is to interact with Rad51 . However , the phosphorylaTION pattern is not altered by specifically disrupting the Rad52-Rad51 interaction (Figure 5E) , suggesting that this interaction , per se , is not required for PHOSphorylation to occur . We , therefore , favor the notion that the C terminus contains PHOSphorylation targets that function to regulate the interaction between Rad52 and Rad51 . Figure 6 summarizes our interpretation of the pattern of electrophoretic mobilities of Rad52 protein . Unmodified Rad52 translated from the fourth and fifth start codons results in the 54 kDa protein band , which is an unresolved doublet , whereas unmodified Rad52 translated from the third start codon migrates more slowly at 57 kDa (Figure 6A) . In G1-arrested wild-type cells , three Rad52 protein bands are detected and they are the result of a combination of unmodified Rad52 species together with a first PHOSphorylation event ( Figure 6B ) . We suggest that the 57 kDa protein band is a combination of unmodified Rad52 expressed from the third start codon together with additional protein species corresponding to PHOSphorylated Rad52 translated from the fourth and fifth start codons ( Figure 6B , 4* and 5* ) . Similarly , PHOSphorylation of Rad52 translated from the third start codon results in a protein band with slower electrophoretic mobility ( 60 kDa ) ( Figure 6B , 3* ) . Next , we explain the additional 63 kDa Rad52 protein band by a second PHOSphorylation event occurring in a cell cycle -dependent manner during S phase ( Figure 5B ; Figure 6C , 3* becoming 3** ) . This cell cycle regulated PHOSphorylation event also shifts the intensity of the other species giving rise to 4** and 5** . Thus , we conclude that the subSTRates for the second modification are the previously PHOSphorylated Rad52 species . A possible role for PHOSphorylation of Rad52 protein may be in the cellular response to DNA damage , where Rad52 relocalizes from a diffuse nuclear distribution to distinct foci at the sites of DSBs ( 41,52 ) . In fact the appearance of spontaneous Rad52 foci coincides with the cell cycle -dependent phosphorylaTION [Figure 5C and ( 43 ) ] , suggesting that some aspect of Rad52 focus formation is regulated by PHOSphorylation . Rad52 PHOSphorylation is not affected in a mec1Delta tel1Delta double mutant , where the two major DNA damage responding kinases are absent ( data not shown ) . This is consistent with the observation that Rad52 PHOSphorylation is not increased after gamma-irradiation ( data not shown ) . Perhaps the first phosphorylaTION event that persists throughout the cell cycle is a negative regulator of Rad52 foci and the cell cycle regulated PHOSphorylation promotes focus formation for homologous recombination .