TI - Rad52 is PHOSphorylated in a cell cycle -independent and cell cycle -dependent manner . AB - Some of the multiple Rad52 protein bands observed for the wild-type strain are accounted for by the promiscuous choice of start codons . For example , the 54 kDa protein band jointly represents Rad52 translated from the fourth and fifth start codons since it is not detected in E24Stop M38A M40A , which can only produce protein from the third start codon ( Figure 4 , lane 9 ) . This is further supported by the observation that the 54 kDa band is present in both M38A and M40A where the fourth and fifth start codons are removed individually ( Figure 4 , lanes 5 and 6 ) . In addition , mutant strains E36Stop , M34A and FS3-4 ( Figure 4 , lanes 3 , 4 and 7 ) , which can express Rad52 protein only from the fourth and fifth start codons , produce a 57 kDa band in addition to the expected 54 kDa band . The presence of the 57 kDa bands suggests that these Rad52 species are post-translationally modified . Furthermore , multiple bands are produced in mutants where Rad52 translation can only initiate from a single start codon ( FS4-5 , E24Stop M38A M40A and E24Stop M34A M38A ) ( Figure 4 , lanes 8-10 ) . To unequivocally determine the mobility of unmodified protein , the yeast RAD52 gene was expressed in E.coli from the third start codon to compare its migration pattern to that of wild-type yeast protein . Rad52 expressed in E.coli produces a single band that co-migrates with the 57 kDa protein band present in yeast extracts of wild-type cells (Figure 5A) . G1-arrested yeast cells expressing Rad52 only from the third start site (E24Stop M38A M40A) produce 57 and 60 kDa bands ( Figure 5B , 0 min ) . When these cells are released into S phase , an additional band appears ( 63 kDa ) ( Figure 5B , 45 min ) , similar to what we previously showed for wild-type Rad52 ( 43 ) . Together these observations show that post-translational modification is responsible for several of the Rad52 species observed . To determine whether the post-translational modification is PHOSphorylation , protein extracts from G1-arrested wild-type cells were treated with lambda-phosphatase . The multiple Rad52 species collapse into two distinct protein bands that co-migrate with the 54 and 57 kDa protein bands from untreated wild-type cells ( Figure 5C , 0 min ) . The 63 kDa cell cycle specific band also disappears after lambda-phosphatase treatment ( Figure 5C , 60 min ) . Finally , lambda-phosphatase treatment of protein extracts from E24Stop M38A M40A , which can only produce Rad52 protein from the third start codon , merges the different protein species into a single 57 kDa Rad52 band ( data not shown ) . Rad52 dephosphorylaTION also occurs after treatment with protein phosphatase 1 , which is specific towards phosPHOserines and phosPHOthreonines , but not after T-cell protein tyrosine phosphatase treatment , a PHOSphotyrosine specific phosphatase ( data not shown ) . In summary , these results demonstrate that Rad52 is PHOSphorylated at serine and/or threonine residues in both a cell cycle -independent and cell cycle -dependent manner .