Stable interactions between DNA polymerase δ catalytic and structural subunits are essential for efficient DNA repair
Introduction
Central to the chromosomal DNA replication apparatus in eukaryotic cells are three essential DNA polymerases, namely DNA polymerase α, δ and ɛ [1]. The primase subunit of the Pol α/primase complex initiates synthesis by oligomerizing short RNA primers on both leading and lagging strands. These primers are initially extended by Pol α and subsequently transferred to processive polymerases Pol ɛ and Pol δ. In contrast to the quite obvious role of Pol α, the specific roles for Pol ɛ and Pol δ in DNA replication were much debated for years. However, recent evidence strongly supports a model of the replication fork wherein the leading and the lagging strand templates are primarily copied by Pol ɛ and Pol δ, respectively [2], [3].
Pol α, Pol δ and Pol ɛ belong to the B class of DNA polymerases [4] and their catalytic subunits are phylogenetically related and conserved from yeast to humans [5], [6]. Similarly, among the different accessory subunits of the replicative Pols, only the so-called B subunit [7], is clearly conserved in eukaryotic organisms [8]. Interestingly, Pol α, Pol δ and Pol ɛ share unifying features of their subunit organization that again underlines their evolutionary relationship. Indeed, the sequence conservation of the catalytic subunit extends past the polymerase fold to a cysteine-rich C-terminal domain (CTD) that contains two zinc fingers. A large body of experimental evidence has highlighted that this CTD interacts with the B subunit and that this interaction is of functional importance in Pol α [9], Pol δ [10], [11] and Pol ɛ [12]. Recently, a 3D architecture of Pol α has been published that allows a clearer overall picture of the catalytic and B subunit interactions for all three replicative polymerases [13]. An interesting conclusion drawn from this structural analysis is that it defines the C-terminal region of the replicative Pols α, δ and ɛ as an independently folded protein domain, separate from the polymerase fold.
DNA polymerase δ from Saccharomyces cerevisiae, has three subunits: Pol3/Cdc2, the catalytic subunit of 125 kDa that contains the polymerase and 3′ to 5′ exonuclease active site domains; Pol31/Hys2, the essential structural B subunit of 55 kDa; Pol32, the additional C subunit of 40 kDa [14]. The enzymes from Schizosaccharomyces pombe and humans have an additional small fourth subunit that functions to stabilize the complex [15], [16], [17]. The enzymes from the three different sources show roughly similar structure characteristics [7] and contain one of each of the subunits, i.e., they are a monomeric catalytic complex [18], [19]. The catalytic and the B subunits form a stable complex (as discussed above), to which the C subunit is tethered solely via interactions with the B subunit [14], [20]. The interaction between the B subunit and the C subunit of Pol δ participates to the functional integrity of the whole complex. Indeed, while POL32 is dispensable for growth in S. cerevisiae, the POL32 deletion mutant nevertheless shows poor growth and synthetic lethality with numerous genes that function in DNA metabolism [14], [21]. In addition, the orthologous S. pombe Cdc27 gene is essential for growth [18], [22]. In both S. cerevisiae and S. pombe, the C subunit interacts with the B subunit via its N-terminal part, whereas its C-terminal part contains a conserved PCNA-binding motif and a motif that mediates interaction with Pol α [18], [23], [24]. It is noteworthy that deletion of the POL32 N-terminal end exhibits the same phenotype than the complete deletion [24]. Using two-hybrid screening, the human C subunit, p66, has been shown to contain p50 (the human Pol δ B subunit) and PCNA binding domains within the 144 N-terminal and 20 C-terminal amino acids, respectively [25]. Recently, the crystal structure of the complex between p50 and the 144 amino acids N-terminal domain of p66 of human Pol δ has been reported [26]. It appears that p66 makes an intricate complex with p50 and has a significantly large surface area buried at the heterodimer interface. Interestingly, in this study, it is noted that in spite of the formation of a very tight complex between p50 and p66 subunits, the residues involved in dimerization correspond to poorly conserved regions among eukaryotic sequences. The analysis of the secondary structure elements of p50 and p66 reveals, however, that this complex is likely to share the same overall fold with its orthologs [26].
The interaction between the Pol δ subunits plays a crucial role in the stability of the Pol δ complex and allows full proficiency of Pol δ in DNA replication. Interestingly, essential functions of Pol δ in DNA repair are even more sensitive to defective interactions within the complex. This observation comes essentially from the study of Pol δ mutants in S. cerevisiae. The pol3-13 mutation leads to the substitution of a Pol3 cysteine at position 1074 by a serine (C1074S). Pol3 contains a CTD with two zinc fingers, ZnF1 and ZnF2. Residue C1074 is in the last ZnF2 cysteine. Two-hybrid studies have shown that the mutated C1074S ZnF2 module can no longer interact with Pol31 [11]. In vivo, the pol3-13 allele confers thermosensitivity, but at the permissive temperature of 30 °C, it confers UV and γ-rays sensitivity and almost a complete defect in both γ- and UV-induced recombination and UV-induced mutagenesis [10]. Similarly, Pol32 interacts with Pol31 but also with Pol1, the catalytic subunit of Pol α [24], [27] and the translesion DNA polymerase Rev1 [28]. Deletion of POL32 is viable but confers UV sensitivity and abolishes UV-induced mutagenesis [29]. In addition, break-induced replication (BIR) repair is seriously impaired in the pol32Δ mutant and gives rise to aberrant double-strand break (DSB) repair resulting in half crossovers [30], [31], [32]. Interestingly, Rev1 is required for UV-induced mutagenesis [33] while Pol α is involved in BIR [31]. Thus, Pol32 interaction with Rev1 and Pol α could have provided a clear molecular basis to understand the role of Pol32 in UV-induced mutagenesis and BIR. However, the deletion of neither the Pol1 or Rev1 interacting domains of Pol32 leads to defects in the corresponding DNA repair pathway [24], [31]. By contrast, the essential functions of Pol32 in DNA repair are comprised in a small amino-terminal domain of Pol32, not much larger than that necessary to specify the interaction with Pol31, suggesting that the Pol32 subunit mediates its role in UV-induced mutagenesis and BIR through the Pol δ complex [24], [31].
In our previous studies, we described the pol3-ct allele that changes a Leu codon to a stop codon at position +1094 in S. cerevisiae resulting in the loss of the four last C-terminal amino acids (LSKW) of Pol3 [34], [35]. Yeast cells carrying this allele show no obvious defects in genome replication since they are neither thermosensitive (ts) nor mutators and they do not display high levels of spontaneous recombination. In addition, they are not sensitive to UV and γ-rays irradiation. We found, however, that the pol3-ct mutant, although repairing DSB as efficiently as WT, yields shorter recombination intermediates in both meiosis and mitosis. This interesting phenotype of the pol3-ct mutant implies that the role of Pol δ during HR is not restricted to gap filling but participates as well in recombination intermediates extension.
The phenotype associated with the pol3-ct allele raises the attractive possibility that in the pol3-ct mutant, Pol δ fails to interact with a protein involved in recombination intermediates extension. However, in our present study, we provide genetic and structural evidence showing that pol3-ct leads to a destabilized interaction between Pol3 CTD and Pol31. Therefore, we reach the alternative conclusion that DNA repair synthesis is clearly dependent on Pol δ complex stability.
Section snippets
Yeast strains, plasmids and media
All media were prepared as previously described [36]. Strains used in the present study are isogenic derivatives of W303-1A [37] and listed in Supplementary Table S1.
Yeast two-hybrid assay
Strain CTY10-5d (MATa, ade2-101, his3Δ200, leu2Δ1, trp1Δ901, gal4, gal80, URA3::lexAop-lacZ) was used for two-hybrid analysis. Pol31 was fused in frame to the lexA binding domain in plasmid pBTM116, while the Pol3 C-terminal amino acids 1032-1097 were fused to the Gal4 activating domain of pACT2 to yield plasmid pACT2-Pol3-ZnF2 [11]
Loss of the last four amino acids at the C-terminal end of Pol3 impairs the interaction with Pol31
While the pol3-ct mutant shows defects in HR and BIR, it does not display any noticeable dysfunction during genome replication, suggesting that Pol δ works efficiently in this mutant. However, Sanchez Garcia and colleagues demonstrated that the C-terminal end of Pol3 is responsible for a direct interaction with Pol31 [11]. Interestingly, they found that deleting the last 13 amino acids of Pol3 disrupts this interaction. Therefore, we used their two-hybrid assay to determine the role of the last
Pol δ stability is impaired in the pol3-ct mutant
pol3-ct has proven to be a useful allele of the essential POL3 gene to study the role of Pol δ in gene conversion and BIR [32], [34], [35]. The present study of pol3-ct sheds light on the role of the C-terminal end of Pol3 in Pol δ stability and allows comprehending the role of Pol δ in DNA repair. We found that the C-terminal end of Pol3 missing the last four LSKW amino acids poorly interacts with Pol31 in a yeast two-hybrid assay. This result is confirmed by the observation that Pol3 ZnF2
Funding
This work was supported by two grants from the Agence Nationale de la Recherche (ANR-07-BLAN-0350-01 and ANR-09-PIRI-0015-03), by INCa/Canceropole IdF and by the CEA and the CNRS. The funders had no role, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests
The authors have declared that no competing interests exist.
Acknowledgements
We thank Dr. Stuart MacNeill for his generous gift of the strain and plasmids used in the yeast two-hybrid assay. We thank Dr. Akio Sugino for his generous gift of yeast strains YNN101 (hys2-2) and YNN102 (pol32::LEU2) and Dr. Dana Branzei for her generous gift of yeast strain KSH542 (hys2-1). We are grateful to S. Boiteux, X. Veaute and E. Coïc for their support and fruitful discussions.
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These authors contributed equally to this work.