Requirement of RAD5 and MMS2 for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae

Abstract

UV lesions in the template strand block the DNA replication machinery. Genetic studies of the yeast Saccharomyces cerevisiae have indicated the requirement of the Rad6-Rad18 complex, which contains ubiquitin-conjugating and DNA-binding activities, in the error-free and mutagenic modes of damage bypass. Here, we examine the contributions of the REV3, RAD30, RAD5, and MMS2 genes, all of which belong to the RAD6 epistasis group, to the postreplication repair of UV-damaged DNA. Discontinuities, which are formed in DNA strands synthesized from UV-damaged templates, are not repaired in the rad5Delta and mms2Delta mutants, thus indicating the requirement of the Rad5 protein and the Mms2-Ubc13 ubiquitin-conjugating enzyme complex in this repair process. Some discontinuities accumulate in the absence of RAD30-encoded DNA polymerase eta (Poleta) but not in the absence of REV3-encoded DNA Polzeta. We concluded that replication through UV lesions in yeast is mediated by at least three separate Rad6-Rad18-dependent pathways, which include mutagenic translesion synthesis by Polzeta, error-free translesion synthesis by Poleta, and postreplication repair of discontinuities by a Rad5-dependent pathway. We suggest that newly synthesized DNA possessing discontinuities is restored to full size by a "copy choice" type of DNA synthesis which requires Rad5, a DNA-dependent ATPase, and also PCNA and Poldelta. The possible roles of the Rad6-Rad18 and the Mms2-Ubc13 enzyme complexes in Rad5-dependent damage bypass are discussed.

FIG. 1.

Requirement of RAD5 for postreplication repair of UV-damaged DNA. rad1Δ (A) and rad1Δ rad5Δ (B) cells were UV irradiated at 2.5 J/m² and then pulse-labeled with [³H]uracil for 15 min, followed by a 30-min (▵) or 6-h (•) chase in high-uracil medium, prior to conversion to spheroplasts and sedimentation of DNA in alkaline sucrose gradients. Synthesis of normal-sized DNA from unirradiated cells pulse-labeled with [³H]uracil for 15 min was followed by a 6-h chase (○). In the unirradiated rad1Δ and rad1Δ rad5Δ cells, normal-sized DNA reconstituted after a 15-min pulse with [³H]uracil and a 30-min chase. The total sedimentation counts was for cells pulse-labeled for 15 min followed by a 30-min or 6-h chase in high-uracil medium and for the unirradiated cells pulse-labeled for 15 min and then chased for 6 h were ∼11,100, 14,500, and 23,850, respectively.

FIG. 2.

Effect of the rev3Δ and rad30Δ mutations on postreplication repair of UV-damaged DNA. rad1Δ rev3Δ (A), rad1Δ rad5Δ rev3Δ (B), rad1Δ rad30Δ (C), and rad1Δ rad5Δ rad30Δ (D) cells were UV irradiated at 2.5 J/m² and pulse-labeled with [³H]uracil for 15 min followed by a 30-min (▵) or 6-h (•) chase in high-uracil medium. Cells were converted to spheroplasts, and the size of nuclear DNA was examined by sedimentation in alkaline sucrose gradients. Sedimentation patterns of DNA from unirradiated cells pulse-labeled with [³H]uracil for 15 min followed by a 6-h chase (○) are also shown.

FIG. 3.

Epistasis analysis of the mms2Δ mutation. Shown is survival after UV irradiation of wild-type strain EMY74.7, its isogenic mms2Δ derivative strain YMMS2.6, and EMY74.7 strains carrying deletion mutations of different RAD genes constructed by the gene replacement method. Survival curves present results from an average of approximately three experiments for each strain.

FIG. 4.

Requirement of MMS2 for postreplication repair of UV-damaged DNA. rad1Δ mms2Δ (A) and rad1Δ mms2Δ rad5Δ (B) cells were UV irradiated at 2.5 J/m² and then pulse-labeled with [³H]uracil for 15 min, followed by a 30-min (▵) or 6-h (•) chase in high-uracil medium, prior to conversion to spheroplasts and sedimentation of DNAs in alkaline sucrose gradients. Synthesis of normal-sized DNA from unirradiated cells pulse-labeled with [³H]uracil for 15 min followed by a 6-h chase (○) is also shown.

FIG. 5.

Rad6-Rad18-dependent pathways for the replication of UV-damaged DNA in yeast. Whereas Polη primarily carries out error-free translesion synthesis through CPDs and the Rad5-dependent error-free postreplication repair pathway is proposed to primarily promote the bypass of (6-4) dipyrimidine lesions by a copy choice type of DNA synthesis, Polζ contributes to the mutagenic bypass of both these UV lesions. The Mms2-Ubc13 protein complex is proposed to function specifically in the Rad5-dependent postreplication repair pathway. Subsequent to the attachment of ubiquitin to Rad5 and/or associated proteins by the Rad6-Rad18 complex, the Mms2-Ubc13 complex may attach additional ubiquitin moieties to these proteins, and that may be important for the assembly of these repair proteins into the replication machinery.