TI - S.cerevisiae , S.paradoxus , S.uvarum and S.monacensis . . AB - ATG codons in-frame with the third ATG codon in S.cerevisiae . ATG codons out-of-frame with the third ATG codon in S.cerevisiae . Stop codons in-frame with the previous ATG encountered are underlined . * denotes conserved nucleotides . Figure 2 Analysis of the third start codon . Note , RAD52-mRNA initiates 10 nt downstream of start codon 2 ( 15 ) . ( A ) Schematic diagram of M34A , E36Stop and FS3-4 mutant strains . M34A results from a methionine-to-alanine substitution at amino acid 34 . E36Stop results from a stop codon inserted at amino acid 36 . FS3-4 mutant results from a frameshift mutation inserted between the third and fourth ATGs . In FS3-4 , translation initiated upstream of the fourth start codon results in truncation at amino acid 63 . Hence in these mutant strains , Rad52 can only be expressed from the fourth and fifth start codons. ( B ) Schematic diagram of E24Stop M38A M40A mutant having a stop codon at residue 24 , and two methionine-to-alanine substitutions at amino acids 38 and 40 . In this mutant , translation can only start at the third start codon . M , methionine residues indicating the position of the ATG codons . A , alanine residues substitute the methionine amino acids at the indicated positions . Residues in gray are not productive for translation start. ( C ) Survival curves of haploid strains after increasing doses of gamma-ray-induced DNA damage . Black diamond , RAD52 ; open circle , rad52 null ; black circle , M34A ; black square , FS3-4 ; inverted open triangle , E36Stop ; and open diamond , E24Stop M38A M40A. ( D ) Gamma-ray survival after overexpression of the RAD52 and M34A genes from 2u plasmids . Cells carrying 2u plasmids were grown and plated on SC-His medium . Open square , RAD52 transformed with pRS423 ( empty vector ) ; open triangle , M34A transformed with pRS423-M34A ; inverted black triangle , M34A transformed with pRS423 ; and black triangle , M34A transformed with pRS423-RAD52 . Figure 3 Mutational analysis of the fourth and fifth start codons. ( A ) Schematic diagram of M38A and M40A mutants . M38A or M40A mutant strains result from methionine-to-alanine substitutions at amino acid 38 or 40 , respectively . Residues in gray are not productive for translation start. ( B ) Survival curves of haploid strains after exposure to gamma-irradiation . Black diamond , RAD52 ; open circle , rad52 null ; inverted black triangle , M38A ; open triangle , M40A. ( C ) Schematic diagram of E24Stop M34A M38A and FS4-5 mutants . The E24Stop M34A M38A mutant strain has a stop codon at residue 24 and two methionine-to-alanine substitutions at amino acids 34 and 38 . In this triple mutant , translation can only start at the fifth start codon . The FS4-5 mutant results from a frameshift mutation inserted between the fourth and fifth ATGs . In FS4-5 , translation initiated upstream of the fifth start codon results in truncation at amino acid 63 . M , methionine residues indicating the position of the ATG codons . A , alanine residues substitute the methionine amino acids at the indicated positions . Residues in gray are not productive for translation start. ( D ) Survival curves of haploid strains after exposure to gamma-irradiation . Black diamond , RAD52 ; open circle , rad52 null ; black circle , FS4-5 ; and open diamond , E24Stop M34A M38A . Figure 4 Protein analysis of the rad52 mutants that disrupt the third , fourth and fifth start codons . Rad52 was examined by immunoblot analysis using an anti-Rad52 antibody . Lane 1 , rad52 null ; lane 2 , RAD52 ; lane 3 , E36Stop ; lane 4 , M34A ; lane 5 , M38A ; lane 6 , M40A ; lane 7 , FS3-4 ; lane 8 , FS4-5 ; lane 9 , E24Stop M38A M40A ; and lane 10 , E24Stop M34A M38A . Equal amounts of total protein were loaded in each lane on the gel . Approximate molecular weights ( kDa ) corresponding to the observed mobility are indicated . Figure 5 PHOSphorylation of Rad52. ( A ) Immunoblot using anti-Rad52 antibody of RAD52 expressed in yeast and E.coli cells. (A-E) Black arrowheads : . . Rad52 protein bands of different electrophoretic mobilities. ( B and C ) Gray arrowhead : . .cell cycle -dependent phosphorylaTION of Rad52. ( B ) Cell cycle -independent and cell cycle -dependent PHOSphorylation of Rad52 protein in E24Stop M38A M40A . Cells were synchronized in G1 by alpha factor and released into S phase at time zero . Protein extracts were made from arrested cells ( time zero ) and at 45 min following alpha factor release. ( C ) DePHOSphorylation of Rad52 . Wild-type RAD52 cells were synchronized in G1 by alpha factor and released into S phase at time zero . Protein extracts were made from arrested cells ( time zero ) and at 60 min following alpha factor release . lambda , indicates that protein extracts were treated with lambda-phosphatase ; - , indicates no phosphatase treatment. ( D ) Immunoblot analysis of cell cycle synchronized rad52-327delta cells . Protein extracts were made 0 , 30 and 60 min after release from G1-arrested cells . Rad52 was examined by immunoblot analysis using anti-Rad52 antibody (I) . Samples were subjected to FACS analysis to determine DNA content of cells at each time point ( II ) . ( D and E ) Empty arrowhead : . .a protein band non-specifically recognized by the anti-Rad52 antibody also present in the rad52 null. ( E ) Immunoblot of RAD52 and rad52-409-412delta strains . Figure 6 Model to explain the multiple Rad52 protein species. ( A ) RAD52 -encoded mRNA allows protein translation from the third and , by leaky scanning , also from the fourth and fifth start codons ( 3 , 4 and 5 , respectively ) . ( B ) First PHOSphorylation : . .cell cycle -independent . Rad52 species from ( A ) ( 3 , 4 and 5 ) are modified resulting in slower migrating protein species ( 3* , 4* and 5* , respectively ) . ( C ) Second PHOSphorylation : . .cell cycle -dependent . During S phase , some of 3* , 4* and 5* are modified into 3** , 4** and 5** , respectively . Approximate molecular weights ( kDa ) corresponding to the observed mobility are indicated .