Defect of Fe-S cluster binding by DNA polymerase δ in yeast suppresses UV-induced mutagenesis, but enhances DNA polymerase ζ – dependent spontaneous mutagenesis

Abstract

Eukaryotic genomes are duplicated by a complex machinery, utilizing high fidelity replicative B-family DNA polymerases (pols) α, δ and ε. Specialized error-prone pol ζ, the fourth B-family member, is recruited when DNA synthesis by the accurate trio is impeded by replication stress or DNA damage. The damage tolerance mechanism dependent on pol ζ prevents DNA/genome instability and cell death at the expense of increased mutation rates. The pol switches occurring during this specialized replication are not fully understood. The loss of pol ζ results in the absence of induced mutagenesis and suppression of spontaneous mutagenesis. Disruption of the Fe-S cluster motif that abolish the interaction of the C-terminal domain (CTD) of the catalytic subunit of pol ζ with its accessory subunits, which are shared with pol δ, leads to a similar defect in induced mutagenesis. Intriguingly, the pol3-13 mutation that affects the Fe-S cluster in the CTD of the catalytic subunit of pol δ also leads to defective induced mutagenesis, suggesting the possibility that Fe-S clusters are essential for the pol switches during replication of damaged DNA. We confirmed that yeast strains with the pol3-13 mutation are UV-sensitive and defective in UV-induced mutagenesis. However, they have increased spontaneous mutation rates. We found that this increase is dependent on functional pol ζ. In the pol3-13 mutant strain with defective pol δ, there is a sharp increase in transversions and complex mutations, which require functional pol ζ, and an increase in the occurrence of large deletions, whose size is controlled by pol ζ. Therefore, the pol3-13 mutation abrogates pol ζ-dependent induced mutagenesis, but allows for pol ζ recruitment for the generation of spontaneous mutations and prevention of larger deletions. These results reveal differential control of the two major types of pol ζ-dependent mutagenesis by the Fe-S cluster present in replicative pol δ.

Introduction

Mutagen-induced lesions in DNA that escape repair [1], [2] can block cell division. This occurs when replicative DNA polymerases cannot incorporate nucleotides across from the damaged sites, leading to replication fork collapse. This collapse can be prevented by DNA damage tolerance mechanisms that allow for the completion of genomic replication [3], [4]. The DNA damage tolerance pathways include mutation-avoiding recombinational and fork reversal mechanisms, and mutation-prone translesion synthesis (TLS) [2], [4], [5], [6], [7], [8].

Mutagenic TLS processes are responsible for most environmentally induced cancers [9], [10], [11]. During TLS there is a switch from accurate, replicative DNA pols to specialized DNA pols. These specialized pols incorporate nucleotides across from the damaged/altered base(s) and then extend from the resulting aberrant termini [12], [13], [14]. Each TLS event can be broken down into three major steps. First, there is the insertion of nucleotides across from the altered base(s) by specialized polymerases [13]. This is typically accomplished by the low fidelity Y-family pols η, κ, ι, and Rev1 [15], [16], and sometimes by X-, A-, or B-family DNA pols, or a member of archaeal-eukaryotic primase family, PrimPol [6], [17], [18]. In the second step, the inserter pol itself or another pol extends the aberrant primer terminus. The efficient bypass and extension of such termini are incompatible with proofreading [19], [20], and as such the participating pols do not have this activity. If an error (inserting a base that does not match the original sequence) was made during bypass, this extension allows the altered sequence to remain in the new DNA strand sequence, leading to a mutation. Most commonly, the extension step in eukaryotes is accomplished by the error-prone B-family pol ζ [5], [9], [17], [21]. In the third step, the resumption of “normal” synthesis downstream of the lesion on the undamaged template occurs. This synthesis can be done by either pol ζ itself up to a new replication restart site, or by switching back to replicative polymerases [6], [22], [23], [24]. The replication fork can restart downstream to allow for continuation of replication as this multistep TLS process of lesion bypass occurs [22]. Filling the gap between the site of bypass and replication restart is another mutagenic threat, because of the longer lifetime of chemically unstable ssDNA templates [25] and the use of less accurate DNA pols [23].

Pol ζ is responsible for most induced point mutations and roughly half of all spontaneous mutations [14], [26]. Pol ζ has relatively low fidelity, and produces a characteristic mutational signature [27]. However, this signature was determined with preparations of yeast pol ζ with incorrect stoichiometry, (mostly Rev3/Rev7 subunits [28]) and with non-physiological equimolar concentrations of nucleotides, which can strongly alter a spectrum of pol mutations [29]. Nevertheless, remnants of this signature can be found in mutation spectra in vivo [2,23,30,31]. One of the features of the signature, complex mutations, are defined as sequence changes of a short stretch of nucleotides and are attributed to template switches [32]. Loss of the catalytic subunit of pol ζ results in decreased spontaneous mutation rates, but elevated rates of large deletions [31], [32] and gross chromosomal abnormalities [33].

Mutation-prone TLS during replication is not necessarily connected to DNA damage. Similar processes, called DRIM (Defective Replisome Induced Mutagenesis), occur in yeast strains with mutant replicative polymerases that have attenuated activity [30], [31], [34], [35], in strains with defects of pol subunits or accessory factors [36], [37], at unusual DNA structures [32], [38], or when nucleotide pools are perturbed [31], [39]. It is generally believed [30] that the cellular machinery for damage-induced and DRIM-like events are the same, due to their common genetic control and dependence on a complex of proteins comprised of replicative pols, TLS pol ζ, Rev1, and monoubiquitylated proliferating cell nuclear antigen (PCNA) [5], [14], [40], [41].

The mechanisms of switches between DNA polymerases responsible for mutations are not well understood. The recent intriguing finding that the subunits POLD2 and POLD3 (Pol31 and Pol32 in yeast) are shared by major replicative pol δ and pol ζ (reviewed in [28]) may be a clue for the mechanism [42]. The Fe-S cluster in the C-terminal part of the catalytic subunits of these pols [43] plays a role in the interaction with POLD2 (Pol31 in yeast) [42], [44]. We proposed that the Fe-S cluster in the catalytic subunit of pol δ plays an additional role in the DNA pol switch [24], because yeast strains bearing allele pol3-13, encoding for a variant of Pol3 with one of the cysteines (Cys 1074) coordinating the Fe-S cluster changed to serine, were reported to have a defect in induced mutagenesis [45]. This allele has amazing pleiotropic effects and a wide spectrum of genetic interactions [45], [46]. Yeast pol3-13 strains are temperature sensitive, with no growth at 37 °C. At 22 °C they grow well, but have elevated spontaneous mutagenesis and recombination. The pol3-13 allele is synthetic lethal with the pol32-Δ mutation, leading to the absence of Pol32, the third subunit of yeast pol δ and the fourth subunit of pol ζ. The latter two phenotypes suggest that pol3-13 leads to decreased stability of pol δ. The pol3-13 mutation is synthetic lethal with mutations affecting the synthesis of Fe-S clusters [46] and the complex pathways of biosynthesis of the Fe-S cofactor (e.g. mms19, nbp35, tah18 and others) [47], [48]. It is also lethal with mutations in homologous recombination pathways. Finally, it is lethal in combination with some mutations affecting signal transduction, transcription, and nuclear transport, consistent with a prominent role for pol δ in DNA metabolism.

In this work, we examined induced and spontaneous mutagenesis in pol3-13 mutant yeast strains and its dependence on the REV3 gene, encoding the catalytic subunit of pol ζ. We confirmed that the defect in coordination of the Fe-S cluster in pol δ conferred by the pol3-13 mutation abolishes UV-mutagenesis, but found that it elevates the incidence of spontaneous Rev3-dependent base-substitution mutations and deletions. The results suggest that, unexpectedly, pol δ differentially regulates pol ζ transactions in induced versus spontaneous mutagenesis.