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. 2006 Oct;26(20):7783-90.
doi: 10.1128/MCB.01260-06. Epub 2006 Aug 14.

Mms2-Ubc13-dependent and -independent roles of Rad5 ubiquitin ligase in postreplication repair and translesion DNA synthesis in Saccharomyces cerevisiae

Venkateswarlu Gangavarapu  1 , Lajos Haracska, Ildiko Unk, Robert E Johnson, Satya Prakash, Louise Prakash
Affiliations

Mms2-Ubc13-dependent and -independent roles of Rad5 ubiquitin ligase in postreplication repair and translesion DNA synthesis in Saccharomyces cerevisiae

Venkateswarlu Gangavarapu et al. Mol Cell Biol. 2006 Oct.

Abstract

The Rad6-Rad18 ubiquitin-conjugating enzyme complex of Saccharomyces cerevisiae promotes replication through DNA lesions via three separate pathways that include translesion synthesis (TLS) by DNA polymerases eta and zeta and postreplicational repair (PRR) of discontinuities that form in the newly synthesized DNA opposite from DNA lesions, mediated by the Mms2-Ubc13 ubiquitin-conjugating enzyme and Rad5. Rad5 is an SWI/SNF family ATPase, and additionally, it functions as a ubiquitin ligase in the ubiquitin conjugation reaction. To decipher the roles of these Rad5 activities in lesion bypass, here we examine the effects of mutations in the Rad5 ATPase and ubiquitin ligase domains on the PRR of UV-damaged DNA and on UV-induced mutagenesis. Even though the ATPase-defective mutation confers only a modest degree of UV sensitivity whereas the ubiquitin ligase mutation causes a high degree of UV sensitivity, we find that both of these mutations produce the same high level of PRR defect as that conferred by the highly UV-sensitive rad5Delta mutation. From these studies, we infer a requirement of the Rad5 ATPase and ubiquitin ligase activities in PRR, and based upon the effects of different rad5 mutations on UV mutagenesis, we suggest a role for Rad5 in affecting the efficiency of lesion bypass by the TLS polymerases. In contrast to the role of Rad5 in PRR, however, where its function is coupled with that of Mms2-Ubc13, Rad5 function in TLS would be largely independent of this ubiquitin-conjugating enzyme complex.

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Figures

FIG. 1.
FIG. 1.
Purity and ATPase activity of wild-type and mutant Rad5 proteins. (A) Schematic representation of Rad5. The highly conserved DE residues at positions 681 and 682 in Rad5 that correspond to conserved ATPase active-site residues and the CC residues at positions 914 and 917 in the C3HC4 ubiquitin ligase motif are indicated by asterisks. (B) Purified wild-type Rad5 and mutant DE681,682AA proteins were analyzed on a 6% denaturing polyacrylamide gel and stained with Coomassie blue. The position of the purified proteins is indicated by an arrow. Lane 1, molecular mass standards; lane 2, 0.5 μg of wild-type Rad5 protein; lane 3, 0.5 μg of Rad5 DE681,682AA. (C) ATPase activity of Rad5 proteins. Purified Rad5 (lanes 2 and 3) and Rad5 DE681,682AA (lane 4) proteins (50 ng) were incubated in 10-μl reaction mixtures containing 20 mM Tris-HCl, pH 7.0, 20 mM KCl, 2 mM MgCl2, 100 μg/ml bovine serum albumin, 1 mM dithiothreitol, and 0.5 mM [γ-32P]ATP in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 200 ng of single-stranded M13 (ssM13) DNA for 5 min at 30°C. ATPase activity was monitored by thin-layer chromatography on polyethyleneimine-cellulose and visualized by autoradiography. NP, no Rad5 protein was added.
FIG. 2.
FIG. 2.
UV sensitivity of rad5 mutations. Survival curves after UV irradiation of wild-type strain EMY74.7 (•), its isogenic rad5Δ (○), mms2Δ (▵), and ubc13Δ (▴) derivatives, and the rad5Δ strain carrying the ATPase-defective DE681,682AA mutation (▪) or the ubiquitin ligase-defective CC914,917AA mutation (□) in the RAD5 gene. Survival curve results represent the averages of at least three experiments.
FIG. 3.
FIG. 3.
Requirement of Rad5 ATPase and ubiquitin ligase activities for postreplication repair of UV-damaged DNA. Sedimentation in alkaline sucrose gradients of nuclear DNA from cells incubated for different periods following UV irradiation is shown. The rad1Δ (A), rad1Δ rad5Δ (B), rad1Δ rad5 CC914,917AA (C), and rad1Δ rad5 DE681,682AA (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 (▵) or 6-h (•) chase in high-uracil medium. The synthesis of normal-sized DNA from unirradiated cells pulse-labeled with [3H]uracil for 15 min was followed by a 6-h chase (○).
FIG. 4.
FIG. 4.
Requirement of Rad5 but not of its ATPase or ubiquitin ligase activities for UV reversion at arg4-17. Cells grown to mid-logarithmic phase in synthetic medium, to which the appropriate supplements for growth of the particular strain had been added, were washed and sonicated to disperse clumps. Cell suspensions were plated on the appropriate synthetic medium and UV irradiated. Colonies were counted after 3 to 5 days of incubation in the dark for viability and mutagenesis determinations. ub, ubiquitin.
FIG. 5.
FIG. 5.
Mms2-Ubc13-dependent and -independent roles of Rad5 in lesion bypass. (Left) Rad5 functions in PRR as a ubiquitin (Ub) ligase in conjunction with Mms2-Ubc13. As a ubiquitin ligase, Rad5 promotes the polyubiquitylation of PCNA at lysine 164 by binding PCNA as well as the Mms-Ubc13 complex and thereby activating the PRR pathway. Also shown is the requirement of Rad5 ATPase in PRR. (Right) Rad5 function in TLS does not depend upon Mms2-Ubc13. Rad5 is proposed to modulate TLS in two separate ways: one dependent upon its ubiquitin ligase function and the other independent of it. As a ubiquitin ligase, Rad5 could affect Rad6-Rad18-dependent ubiquitylation of TLS Pols; in addition, by binding to both the inserter and the extender Pols, Rad5 could coordinate the action of the two Pols via a direct structural involvement.
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