Saccharomyces cerevisiae RAD5-encoded DNA repair protein contains DNA helicase and zinc-binding sequence motifs and affects the stability of simple repetitive sequences in the genome.

Abstract

radS (rev2) mutants of Saccharomyces cerevisiae are sensitive to UV light and other DNA-damaging agents, and RADS is in the RAD6 epistasis group of DNA repair genes. To unambiguously define the function of R4DS, we have cloned the RAD5 gene, determined the effects of the radS deletion mutation on DNA repair, DNA damage-induced mutagenesis, and other cellular processes, and analyzed the sequence of RAD5-encoded protein. Our genetic studies indicate that R4DS functions primarily with R4D18 in error-free postreplication repair. We also show that R4DS affects the rate of instability of poly(GT) repeat sequences. Genomic poly(GT) sequences normally change length at a rate of about 10-4; this rate is approximately 10-fold lower in the radS deletion mutant than in the corresponding isogenic wild-type strain. RADS encodes a protein of 1,169 amino acids of M, 134,000, and it contains several interesting sequence motifs. All seven conserved domains found associated with DNA helicases are present in RAD5. RAD5 also contains a cysteine-rich sequence motif that resembles the corresponding sequences found in 11 other proteins, including those encoded by the DNA repair gene RA4D8 and the RAGI gene required for immunoglobin gene arrangement. A leucine zipper motif preceded by a basic region is also present in RAD5. The cysteine-rich region may coordinate the binding of zinc; this region and the basic segment might constitute distinct DNA-binding domains in RAD5. Possible roles of RAD5 putative ATPase/DNA helicase activity in DNA repair and in the maintenance of wild-type rates of instability of simple repetitive sequences are discussed.DNA containing pyrimidine dimers and other types of UV damage is a poor template for the replication machinery. Since DNA polymerases cannot copy past such damage, a gap ensues in the newly synthesized strand across from the damage site. This gap can be filled in by several different postreplication repair processes. Genetic evidence from Saccharomyces cerevisiae suggests that the majority of gaps are filled in an error-free manner in which information for gap filling is obtained from the undamaged strand in the sister duplex (45). Either recombinational or nonrecombinational mechanisms may be utilized. In recombination, the strand from the sister duplex fills in the gap; a nonrecombinational mechanism might entail a copy choice type of DNA synthesis in which blocked DNA polymerase switches from the damaged template, carries out translesion DNA synthesis by copying the undamaged strand in the sister duplex, and then switches back to the damaged template after clearing the lesion. Under certain circumstances, gap filling can occur by mutagenic bypass of the lesion.To elucidate the mechanisms employed by eukaryotic cells to fill in the gap across from the lesion, we have been studying the genes that function in nonmutagenic and mutagenic postreplication repair pathways in S. cerevisiae. Several of these genes have been cloned, and in some cases, their protein products have been purified and their b...

A mutation in EXO1 defines separable roles in DNA mismatch repair and post-replication repair

“…RAD5 deficiency results in higher sensitivity to various types of DNA damage than deletion of either MMS2 or UBC13 [33]. Furthermore, rad5Δ mutants, relative to other PRR mutants, are highly sensitive to ionizing radiation, have elevated rates of spontaneous mitotic recombination, higher rates of gross chromosomal rearrangements, paradoxically increased stability of simple repetitive sequences and higher end-joining activity in a plasmid gap repair [53][54][55][56]. Similar to our findings, Chen et.…”

The error-free component of the RAD6/RAD18 DNA damage tolerance pathway of budding yeast employs sister-strand recombination

“…interaction between rad5 and rad6 mutations (37) together with the absence of any marked effect of rad5 mutations on induced mutagenesis (38). It is also supported by the discovery that the Rad5͞Ubc13͞Mms2 complex polyubiquitinates lysine 164 in proliferating cell nuclear antigen by conjugating ubiquitin to lysine 63 of ubiquitin itself (14), whereas monoubiquitination of lysine 164 promotes translesion replication (15) and polyubiquitination promotes the RAD6͞RAD18-dependent error-free process of DNA damage tolerance (14).…”

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

“…Hence, at least two E2-E3 complexes, namely Rad6-Rad18 and Mms2-Ubc13-Rad5, are required for DDT in yeast. In addition, RAD5 has been reported to promote instability of simple repetitive sequences [57] and to inhibit non-homologous end-joining of DSBs [59]. Indeed, Rad5 is involved in double-strand break repair independent of its ubiquitination activity [60].…”

“…The cognate E3 for Mms2-Ubc13 turns out to be Rad5, another RING-finger protein that interacts with both Ubc13 and Rad18 [56]. RAD5 encodes a protein with DNA helicase and zinc-binding domains [57] and DNA-dependent ATPase activity [58]. Hence, at least two E2-E3 complexes, namely Rad6-Rad18 and Mms2-Ubc13-Rad5, are required for DDT in yeast.…”