Multiple start codons and phosphorylation result in discrete Rad52 protein species

Adriana Antu nez de Mayolo 1 4 Michael Lisby 1 3 4 Naz Erdeniz 1 2 4 Tanja Thybo 0 1 4 Uffe H. Mortensen 0 1 4 Rodney Rothstein 1 4 0 Center for Microbial Biotechnology , BioCentrum-DTU, Technical University of Denmark , Building 223, DK-2800 Lyngby, Denmark 1 Present address: Adriana Antunez de Mayolo, Sylvester Comprehensive Cancer Center, University of Miami Medical Center , 1550 NW 10th Avenue, Miami, FL 33136, USA 2 Department of Molecular and Medical Genetics, Oregon Health Sciences University , 3181 SW Sam Jackson Park Road, Mail Code L103, Portland, OR 97201, USA 3 Department of Genetics, Institute of Molecular Biology and Physiology, University of Copenhagen , ster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark 4 Department of Genetics & Development, Columbia University Medical Center , 701 West 168th Street, New York, NY 10032, USA The sequence of the Saccharomyces cerevisiae RAD52 gene contains five potential translation start sites and protein-blot analysis typically detects multiple Rad52 species with different electrophoretic mobilities. Here we define the gene products encoded by RAD52. We show that the multiple Rad52 protein species are due to promiscuous choice of start codons as well as post-translational modification. Specifically, Rad52 is phosphorylated both in a cell cycle-independent and in a cell cycle-dependent manner. Furthermore, phosphorylation is dependent on the presence of the Rad52 C terminus, but not dependent on its interaction with Rad51. We also show that the Rad52 protein can be translated from the last three start sites and expression from any one of them is sufficient for spontaneous recombination and the repair of gamma-ray-induced doublestrand breaks. - Saccharomyces cerevisiae RAD52 was initially isolated in a genetic screen for mutants that were sensitive to radiation and to agents known to cause DNA double-strand breaks (DSBs) (1,2). Further analysis showed that rad52 mutant strains display a pleiotropic phenotype, including defects in DNA DSB repair, reduced mitotic and meiotic recombination rates, deficient mating-type switching, elevated mutation rates, increased chromosome loss and low spore viability (38). The importance of RAD52 is underscored by its conservation through evolution in species ranging from yeast to humans (914). Although the sequence of S.cerevisiae RAD52 has been known for more than 20 years (15), the precise gene product(s) have not been defined. This is due to the presence of five potential translation start sites in the RAD52 open reading frame (ORF). Previously, analysis by S1 nuclease digestion of the RAD52-encoded mRNA has shown that a major transcript starts 10 nt downstream of the second ATG triplet in the ORF (15). In accordance with this, most biochemical analyses of Rad52 have employed protein expressed from the third AUG triplet, leaving open the possibility that important aspects of the protein have been overlooked. It is possible that endogenous expression of Rad52 is more complex, as has been shown for other genes with multiple start codons. For example, the SUC2 invertase gene in yeast contains multiple ATG codons, and initiation at the first start site yields a glycosylated, secreted invertase protein. However, when translation initiates from a downstream AUG, the result is an unglycosylated invertase protein that remains in the cell (16). The ribosomal scanning model (1720) proposes a mechanism by which eukaryotic ribosomes select the translation initiation site. Briefly, the 40S ribosomal subunit binds near the free 50 end of an mRNA and migrates through the noncoding region scanning for a translational start site. In yeast and higher eukaryotes, 95% of translation initiates at the AUG triplet near the 50 end (21,22). However, some eukaryotic transcripts have more complex arrangements and the sequence surrounding the AUG codon can either enhance or inhibit initiation of translation at that site. Hence, if the first AUG triplet encountered occurs in a suboptimal sequence recognition context, some 40S subunits will bypass this triplet and initiate translation at a downstream start site by a process termed leaky scanning (2327). Three features near the start codon influence leaky scanning. First, multiple studies have analyzed mRNA sequences and the context surrounding the AUG triplet in many yeast genes to determine whether a consensus sequence exists (22,28,29). From these studies, only the preference for a purine at position 3 is consistent. This preference has been confirmed by mutational analysis of the 50 upstream region of the CYC7 gene fused to the coding region of lacZ (27). In that study, the preferred nucleotide at position 3 was A>G>C>T, with relative protein translational levels 100:94:69:54 (27). In the case of the S.cerevisiae RAD52 gene, the nucleotide at the 3 position for the five ATG codons (from 50 to 30) is C, C, T, A and G, respectively. Second, the length of the 50 mRNA leader sequence also influences leaky scanning (30,31). In yeast, the majority of leader sequences (70%) range from 20 to 60 nt, which is sufficiently long to support initiation of translation (22). Inspection of the RAD52-encoded mRNA by S1 nuclease digestion (15) shows that a major transcript starts 10 nt downstream of the second ATG triplet, thus allowing for a minimum leader sequence of 47 nt upstream of the remaining ATGs. Finally, experimental data from mammalian systems suggest that a stable stemloop structure 1215 nt downstream of a start codon can cause the scanning ribosome to pause with its AUG-recognition center right over the initiator codon, providing more time for the recognition of this start site (32). In the S.cerevisiae RAD52-encoded mRNA, a potential stemloop structure is located downstream of the fifth start codon. This hairpin may influence the preference of the ribosome for the start codons differently as the distance separating the stemloop structure from the third, fourth and fifth start codons is 33, 21 and 15 nt, respectively. Size determination of the Rad52 protein by gelelectrophoresis followed by immunoblot analysis typically detects multiple protein species. The existence of different Rad52 species suggests a promiscuous start codon choice. In addition, some of the multiple protein species could also result from post-translational modification and/or degradation. Here we show that yeast RAD52-encoded mRNA produces Rad52 expressed from three different start sites and that DNA DSB repair is supported by the three protein species. In addition, Rad52 is phosphorylated both in a cell cycle-independent and cell cycle-dependent manner and these modifications require the presence of the Rad52 C terminus. Finally, maintenance of wild-type Rad52 protein levels is important for efficient repair of some types of DNA damage. MATERIALS AND METHODS Genetic methods, yeast strains, site-directed mutagenesis and plasmids Media were prepared as described previously and contain (...truncated)