Mgs1 and Rad18/Rad5/Mms2 are required for survival of Saccharomyces cerevisiae mutants with novel temperature/cold sensitive alleles of the DNA polymerase δ subunit, Pol31

Abstract

Saccharomyces cerevisiae DNA polymerase delta (Polδ) is a heterotrimeric enzyme consisting of Pol3 (the catalytic subunit), Pol31 and Pol32. New pol31 alleles were constructed by introducing mutations into conserved amino acid residues in all 10 identified regions of Pol31. Six novel temperature-sensitive (ts) or cold-sensitive (cs) alleles, carrying mutations in regions III, IV, VII, VIII or IX, conferred a range of defects in the response to replication stress or DNA damage. Deletion of SGS1, RAD52, SRS2, MRC1 or RAD24 had a deleterious effect only in combination with those pol31 alleles that had a phenotype as single mutants, suggesting a requirement for recombination and checkpoint functions in processing the DNA lesions or structures that form as a consequence of replication with a defective Polδ. In contrast, deletion of POL32 negatively affected the growth of almost all pol31 mutants, suggesting an important role for all conserved amino acids of Pol31 in maintaining the integrity of Polδ complex structurally, at least in the absence of the third subunit. Surprisingly, deletions of RAD18 and MGS1 aggravated the temperature sensitivity conferred by most ts or cs alleles and specifically suppressed the hys2-1 and hys2-1-like mutations of POL31. Deletion of RAD5 or MMS2 had an effect on pol31 ts/cs mutants similar to that of RAD18, whereas deletion of RAD30 or REV3 had no effect. We propose that Rad18/Rad5/Mms2 and Mgs1 are required to promote replication when forks are destabilized or stalled due to defects in Polδ. These data are consistent with the biochemical activity of the human Mgs1 orthologue, which binds and stimulates Polδ in vitro. We also demonstrate that Mgs1 interacts physically with Pol31 in vivo. Moreover, regions I and VII of Pol31, which are specifically sensitive to high levels of Mgs1 and PCNA, could be sites of interaction.

Introduction

DNA polymerases carry out a large variety of synthetic transactions during DNA replication, repair and recombination [1], [2]. Polymerases α, δ and ɛ, which are members of the B family of polymerases, are thought to function together at the replication fork to copy genomic DNA in a semi-continuous manner [3]. DNA polymerase delta (Polδ) plays vital roles in eukaryotic cells [4], [5]. In the SV40 in vitro replication system, Polδ is responsible for continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand [6], [7]. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, Polδ is required to complete chromosomal replication [8]. In these model eukaryotes, Polδ likely replicates the lagging strand, but under special circumstances, such as in mutants lacking the Polɛ catalytic domain, Polδ can carry out leading strand synthesis [9], [10], [11], [12]. However, the precise cellular roles of Polδ and Polɛ, and their relationship with one another, are still unclear. Polδ appears to be the major replicative polymerase in eukaryotic cells, and it is the primary polymerase for most DNA repair and recombination pathways, having been implicated in nucleotide excision repair and base excision repair, and in recombination [4]. However, it is not yet clear how Polδ is regulated in these DNA-dependent processes.

Polδ has been purified from several sources. In S. cerevisiae, Polδ is a trimeric enzyme consisting of 125, 55 and 40 kDa polypeptides, comprising the catalytic subunits Pol3, Pol31 and Pol32, respectively [13]. In S. pombe and man, the complex includes a fourth small subunit. Fission yeast Polδ consists of the Pol3, Cdc1, Cdc27 and Cdm1 subunits, and the human enzyme consists of the p125, p50, p68 and p12 subunits [14], [15], [16]. Whereas the first and the second subunits of Polδ in S. cerevisiae which are encoded by the essential genes POL3/CDC2 and POL31/HYS2/SDP5, respectively, are highly conserved in eukaryotes, the third subunit, encoded by the non-essential gene POL32, is extremely divergent at the primary amino acid level.

On the basis of comparative protein sequence analysis, the largest subunits of S. pombe and S. cerevisiae Polδ, which carry out catalytic functions, are likely to be composed of multiple structural domains. The polymerase domain most probably adopts the classical palm–fingers–thumb structure of other B family polymerases. The region on the amino-terminal side of the polymerase domain contains a 3′–5′ exonuclease activity which performs a proofreading function during DNA synthesis. In budding yeast, mutations of conserved amino acids within the exonuclease domain cause a large increase in spontaneous mutation rates [17]. Two putative C4 zinc finger modules are located near the carboxy terminus of the catalytic subunit. Direct interaction between the catalytic subunit of yeast Polδ and a second subunit (Cdc1 in S. pombe or Pol31 in S. cerevisiae) occurs via the more carboxy-terminal zinc finger module, and this region is necessary and sufficient to bind the second subunit in vivo and in vitro [18].

In contrast to S. pombe, in which the gene encoding the third Polδ subunit, Cdc27, is essential for growth, the S. cerevisiae orthologue POL32 is not essential, but a deletion mutant is cold-sensitive (cs) and sensitive to hydroxyurea (HU) and DNA damaging agents [13], [19], [20]. In addition, lethality was observed when the pol32Δ mutation was combined with conditional mutations in POL3, POL31, POL30 (PCNA) or Polɛ [12], [13], [21], [22]. Furthermore, pol32Δ strains are weak antimutators and are defective for damage-induced mutagenesis [13]. Pol32 is required for DNA polymerase zeta (Polζ)-dependent spontaneous mutagenesis and for error-prone DNA synthesis in the presence of DNA damage, suggesting that it is involved in the mutagenic bypass pathway [20], [23], [24], [25]. In addition, Pol32 interacts with Pol31, PCNA, and Pol1 (the large subunit of Polα) [22]. Compared to the fission yeast and human orthologues, only one motif is highly conserved in Pol32, the consensus PCNA-binding motif QXX(L/I)XXFF at the extreme carboxy-terminal region. The binding of Pol32 to Pol1 requires the carboxy-proximal region of Pol32. Surprisingly, it was demonstrated that the essential functions of Pol32 reside in a small amino-terminal domain, not much larger than the amino-terminal 92 amino acids that are required for interaction with Pol31 [22].

The gene encoding the second subunit of Polδ in S. cerevisiae, POL31, was initially identified as the alleles sdp1 and sdp5-1, which are extragenic suppressors of the thermosensitive POL3 mutations pol3-14 and pol3-11, respectively [26]. Later, sdp5-15, which is allelic to sdp5-1, was found to be an extragenic suppressor of pol3-13, a DNA repair-deficient temperature-sensitive allele of POL3 that has a mutation in the cysteine-rich domain [27]. The same gene was isolated as HYS2 in a screen for mutants that exhibit hydroxyurea sensitivity, which yielded the hys2-1 mutant [28]. Finally, the HYS2 (SDP5) gene was identified as encoding one of the small subunits of Polδ and designated POL31 to indicate that it is the second subunit of Polδ in S. cerevisiae [29], [30]. Based on protein sequence comparisons across several eukaryotic species, 10 conserved regions have been identified in Pol31 [31].

Although the functions of the first and the third subunits of S. cerevisiae Polδ have been characterized, there is little information about the second subunit. Thus far, it is known that Pol31 has a structural role in linking Pol3 and Pol32 in the heterotrimeric holoenzyme; however, its exact contributions to Polδ functions are unknown. We previously found that deletion of RAD18, MMS2 or MGS1 suppresses the temperature sensitivity conferred by the hys2-1 mutation, suggesting a functional role for Rad18 or Mgs1 in modulating Polδ functions during DNA replication [32]. We further suggested that the Rad18/Rad5/Mms2 polyubiquitination pathway is important for the completion of replication, perhaps by promoting a template switching mode of DNA synthesis which is detrimental to hys2-1 [33]. These observations prompted us to further study the interplay between Polδ and proteins involved in genome integrity to gain further insights into the roles of Pol31 in replication, repair and recombination.

In this study, we employed site-directed mutagenesis to substitute conserved amino acid residues in the 10 identified regions of Pol31 with alanine and constructed numerous pol31 alleles for genetic analyses. Through initial characterization of the yeast expressing the mutant alleles, we isolated six novel ts and cs mutants of pol31 that exhibit a range of defects in responding to replication stress and DNA damage. Moreover, we examined genetic interactions between each pol31 allele and proteins involved in genome integrity such as Sgs1, Rad52, Srs2, Mrc1, Rad24, Pol32, Mgs1 and PCNA. Surprisingly, deletion of RAD18 or MGS1 has negative effects on the growth of the newly identified pol31 alleles. Furthermore, we found that the rad18Δ and mgs1Δ mutations suppress only those phenotypes that are caused by pol31 mutations affecting amino acids close to the site of the hys2-1 mutation. We have delineated the RAD6-RAD18-pathway by extending our investigation to the effect of deleting RAD30, REV3, RAD5 or MMS2 from our ts/cs mutants. We also demonstrated a physical interaction between Pol31 and Mgs1 in vivo. Based on these results, we discuss the biological significance of the conserved regions and amino acids of Pol31 and their contribution to Polδ functions and interactions.