Requirement of RAD52 group genes for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, replication through DNA lesions is promoted by Rad6-Rad18-dependent processes that include translesion synthesis by DNA polymerases eta and zeta and a Rad5-Mms2-Ubc13-controlled postreplicational repair (PRR) pathway which repairs the discontinuities in the newly synthesized DNA that form opposite from DNA lesions on the template strand. Here, we examine the contributions of the RAD51, RAD52, and RAD54 genes and of the RAD50 and XRS2 genes to the PRR of UV-damaged DNA. We find that deletions of the RAD51, RAD52, and RAD54 genes impair the efficiency of PRR and that almost all of the PRR is inhibited in the absence of both Rad5 and Rad52. We suggest a role for the Rad5 pathway when the lesion is located on the leading strand template and for the Rad52 pathway when the lesion is located on the lagging strand template. We surmise that both of these pathways operate in a nonrecombinational manner, Rad5 by mediating replication fork regression and template switching via its DNA helicase activity and Rad52 via a synthesis-dependent strand annealing mode. In addition, our results suggest a role for the Rad50 and Xrs2 proteins and thereby for the MRX complex in promoting PRR via both the Rad5 and Rad52 pathways.

FIG. 1.

Requirement of RAD51, RAD52, and RAD54 genes for postreplication repair of UV-damaged DNA. Sedimentation in alkaline sucrose gradients of nuclear DNA from cells incubated for different periods following UV irradiation with 3.5 J/m². The rad1Δ (A), rad1Δ rad51Δ (B), rad1Δ rad52Δ (C), and rad1Δ rad54Δ (D) strains were UV irradiated at 3.5 J/m² and then pulse-labeled with [³H]uracil for 15 min, followed by a 30-min (Δ) or 6-h (•) chase in high-uracil medium. Synthesis of normal-size DNA from unirradiated cells pulse-labeled with [³H]uracil for 15 min was followed by a 6-h chase (○). The data for the rad1Δ strain in panel A shown here for comparison are taken from reference .

FIG. 2.

RAD5 and RAD52 control alternate pathways for postreplication repair of UV-damaged DNA. Sedimentation in alkaline sucrose gradients of nuclear DNA from cells incubated for different periods following UV irradiation with 3.5 J/m². The rad1Δ rad5Δ (A) and rad1Δ rad5Δ rad52Δ (B) strains were UV irradiated at 3.5 J/m² and then pulse-labeled with [³H]uracil for 15 min, followed by a 30-min (Δ) or 6-h (•) chase in high-uracil medium. Synthesis of normal-size DNA from unirradiated cells pulse-labeled with [³H]uracil for 15 min was followed by a 6-h chase (○). The data for the rad1Δ rad5Δ strain in panel A shown here for comparison are taken from reference .

FIG. 3.

Involvement of RAD50 and XRS2 in postreplication repair of UV-damaged DNA. Sedimentation in alkaline sucrose gradients of nuclear DNA from cells incubated for different periods following UV irradiation with 3.5 J/m². The rad1Δ rad50Δ (A), rad1Δ xrs2Δ (B), rad1Δ rad50Δ rad5Δ (C), rad1Δ xrs2Δ rad5Δ (D), rad1Δ rad50Δ rad52Δ (E), and rad1Δ xrs2Δ rad52Δ (F) strains were UV irradiated at 3.5 J/m2 and then pulse-labeled with [³H]uracil for 15 min, followed by a 30-min (Δ) or 6-h (•) chase in high-uracil medium. Synthesis of normal-size DNA from unirradiated cells pulse-labeled with [³H]uracil for 15 min was followed by a 6-h chase (○).

FIG. 4.

Epistasis relationships of rad50 and xrs2 with rad5 and rad52. Survival after UV irradiation of wild-type strain EMY74.7 and its isogenic derivatives. Survival curves represent an average for at least three different experiments for each strain. (A) Epistasis of rad50Δ with mms2Δ and with rad5Δ; (B) lack of epistasis of xrs2Δ with rad5Δ and with mms2Δ; (C) increased UV sensitivity of the rad50Δ and xrs2Δ mutations when combined with the rev3Δ or rad30Δ mutations; (D) epistasis of rad50Δ with the rad52Δ mutation and the lack of epistasis between the xrs2Δ and rad52Δ mutations. Error bars represent standard errors.

FIG. 5.

Rad6-Rad18-dependent and Rad51-, Rad52-, and Rad54-dependent pathways for replication of UV-damaged DNA in yeast. It is proposed (see text for details) that Rad5-mediated PRR is restricted to the leading strand and that Rad proteins 51, 52, and 54, which are also likely to involve the other proteins that function with this group, such as Rad55 and Rad57, promote lesion bypass on the lagging strand. Further, it is suggested that both of the PRR pathways utilize nonrecombinational means that involve fork regression and template switching mediated by Rad5 (5) and the SDSA pathway, in which the Rad51-coated ss nucleoprotein filament formed on the strand with the 3′-OH terminus from the gapped region on the lagging strand invades the duplex on the leading strand side, and this is followed by D-loop formation, synthesis, and reannealing reactions (25, 47).