Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
Pol32 is required for Polζ-dependent translesion synthesis and prevents double-strand breaks at the replication fork
Introduction
Stalled replication machinery on the DNA is a critical threat to the cell. This can occur when the polymerase is unable to process beyond a particular point for any reason, such as when DNA damage is encountered that the polymerase cannot replicate past. The cell will die if there is no resolution to this problem, and there are several strategies that the cell may employ to maintain genome integrity. These include cell cycle checkpoints, which respond to DNA damage by halting cell cycle progression [1]. Once DNA damage is sensed, the cell cycle is slowed or stopped so that the lesion may be repaired before the onset of S phase [2]. In this way, the problem of encountering lesions during DNA replication can be minimized. However, this system is not perfect, and since some lesions will persist into S-phase, the cell must have methods to overcome these obstacles or to resolve the stalled replication fork [3]. These are often collectively called damage tolerance pathways, since the lesion is not repaired, but “tolerated” as the cell finds a way to replicate past the damaged DNA. These mechanisms include an exonuclease that processes stalled replication forks and counteracts fork reversal [4], DNA double-strand break (DSB) repair to deal with fork collapse [5] and DNA helicases and topoisomerases to suppress crossovers during DSB repair [6].
Another general damage tolerance mechanism is called DNA postreplication repair (PRR), which is defined as an activity capable of converting DNA damage-induced single-stranded DNA (ssDNA) gaps into continuous high molecular weight double-stranded DNA [7], [8]. It is now clear that the function of this pathway depends on the presence of both Rad6, a ubiquitin-conjugating (Ubc or E2) enzyme [9] and Rad18, a ssDNA binding protein [10] with a RING finger motif characteristic of a ubiquitin ligase (Ubl or E3). These two proteins form a heterodimer [10] that monoubiquitinates proliferating cell nuclear antigen (PCNA) [11], which is absolutely required for the PRR pathway. The PRR pathway is comprised of two parallel subpathways, one error-free and one error-prone [12]. The error-prone or mutagenesis pathway is represented by a translesion synthesis (TLS) polymerase, Polζ, made up of two subunits encoded by REV3 and REV7 [13]. A deoxycytidyl transferase encoded by REV1 is also required for this type of repair [14]. The error-free subpathway includes such proteins as Rad5 [15], Mms2 [16], and Ubc13 [17], [18], which mediate non-mutagenic repair [19]. These three proteins form a E2 (Ubc13-Mms2)–E3 (Rad5) complex [20] that is required for a Lys63-mediated polyubiquitination [18] of PCNA following its monoubiquitination by Rad6-Rad18 [11]. Interestingly, the same Lys residue on PCNA, Lys164, can also be modified by SUMO [11], and it has been reported that PCNA sumoylation may be involved in spontaneous mutagenesis [21] and recruitment of Srs2 to prevent recombination during S-phase [22], [23]. Although it is conceivable that while monoubiquitinated PCNA promotes TLS [21] and Lys63-polyubiquitinated PCNA is required for the error-free mode of bypass [11], questions remain as to what events happen to relay the signal of PCNA modifications to different PRR activities [24].
The involvement of PCNA, a DNA clamp for replicative polymerases, in PRR supports a model in which there is a switch from replication to PRR at sites of replication blocks and suggests the involvement of replicative DNA polymerase(s) in PRR. Polδ is the main replicative polymerase in the eukaryotic cell, and is also thought to be important for many repair activities, including PRR [25], [26]. In the budding yeast Saccharomyces cerevisiae, Polδ is made up of three subunits: Pol3, which is the catalytic subunit and is essential [27], [28]; Pol31, also essential but with a less well understood function, possibly scaffolding [27], [28]; and the non-essential subunit Pol32. Pol32 has been shown to physically interact with itself, the other subunits of Polδ, and PCNA [29], [30] as well as Polα [30]. Cells lacking Pol32 have a number of defects including cold sensitivity for growth [29], [30], [31], reproductive defects characterized by the presence of large budded cells with single mass of duplicated DNA at the mother-bud neck, an increased sensitivity to HU, UV, and DNA methylation damage [29], [30], and a defect in induced mutagenesis [29]. The latter phenotypes imply that Pol32 may be involved in PRR. Since Pol32 is a non-essential subunit of Polδ, we envisioned a role for Pol32 in coordinating or recruiting the necessary proteins for PRR. Our aim in this study was to further characterize the role of Pol32 in DNA repair, especially its involvement in PRR as well as genetic interactions with other relevant DNA metabolism pathways.
Section snippets
Yeast strains and cell culture
The haploid S. cerevisiae strains used in this study are listed in Table 1. DBY747 was originally obtained from Dr. D. Botstein (Stanford University, CA). SJR751 and SJR897 were obtained from Dr. S. Jinks-Robertson (Emory University, Atlanta, GA). HK578 and its isogenic rad9Δ∷HIS3 mutant were received from Dr. H. Klein (New York University, NY). RKY2672 was obtained from Dr. R. Kolodner (University of California, San Diego, CA). Other strains are all isogenic derivatives of the above strains
Pol32 functions within the PRR pathway
With the knowledge that PCNA [11], [46] and Polδ [26] are involved in PRR, we hypothesized that POL32 may be required for PRR as well, probably by serving as a coupling factor between chromosomal replication and lesion bypass. To test this hypothesis, we first created a pol32 rad18 double mutant and compared its UV-sensitivity to that of the corresponding single mutants and the wild type. As shown in Fig. 1A, the pol32 rad18 double mutant is no more sensitive than the rad18 single mutant,
Discussion
POL32 encodes a non-essential subunit of Polδ and is expected to serve two purposes for the Polδ polymerase complex in vivo. One is to act as an accessory component for Polδ to enhance its activity and another is to couple replication to other cellular processes. The former activity of Pol32 has been demonstrated to be a processivity factor, since in the absence of Pol32, the Pol3–Pol31 complex displays poor processivity and stability in vitro, and is characterized by frequent stalls during
Acknowledgments
We wish to thank Renee Brost for technical assistance, Leslie Barbour for useful discussion and several laboratories for providing yeast strains and plasmids. This work was supported by a Canadian Institutes of Health Research operating grant (MOP-38104) to WX.
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