DNA Polymerase δ Is Preferentially Recruited during Homologous Recombination To Promote Heteroduplex DNA Extension

Abstract

DNA polymerases play a central role during homologous recombination (HR), but the identity of the enzyme(s) implicated remains elusive. The pol3-ct allele of the gene encoding the catalytic subunit of DNA polymerase ␦ (Pol␦) has highlighted a role for this polymerase in meiotic HR. We now address the ubiquitous role of Pol␦ during HR in somatic cells. We find that pol3-ct affects gene conversion tract length during mitotic recombination whether the event is initiated by single-strand gaps following UV irradiation or by site-specific double-strand breaks. We show that the pol3-ct effects on gene conversion are completely independent of mismatch repair, indicating that shorter gene conversion tracts in pol3-ct correspond to shorter extensions of primed DNA synthesis. Interestingly, we find that shorter repair tracts do not favor synthesis-dependent strand annealing at the expense of double-strand-break repair. Finally, we show that the DNA polymerases that have been previously suspected to mediate HR repair synthesis (Pol and Pol) do not affect gene conversion during induced HR, including in the pol3-ct background. Our results argue strongly for the preferential recruitment of Pol␦ during HR.Homologous recombination (HR) is a process that allows genetic exchange between DNA sequences sharing homology and leads to gene conversion or crossovers (COs). The ingenuity of this process is underscored by its conservation from bacteria to humans and its implication in a variety of unrelated nuclear processes. HR is implicated in the restart of stalled replication forks (24). It serves in the repair of DNA damage, such as single-strand gaps, double-strand breaks (DSBs) and interstrand cross-links. It is implicated in mating type switching in yeast strains and in the diversification of immunoglobulinvariable genes in vertebrates (6). In meiosis, the primary function of HR is to establish a physical connection between homologous chromosomes to ensure their correct disjunction at the first meiotic division. In addition, meiotic HR contributes to diversity by creating new linkage arrangements between genes or parts of genes (35).In the past few years, it has become evident that these multiple roles of HR are achieved with variations in the process. Two primary models allow for gene conversion as it is observed in different contexts (Fig. 1). In the seminal DSB repair (DSBR) model of Szostak et al. (41), supported mainly by molecular meiotic studies in yeast, the formation of DSBs is followed by exonucleolytic degradation of the 5Ј ends of the broken duplex to expose single-stranded tails with 3Ј termini (40) (Fig. 1). DSB formation and 5Ј-end resection are followed by the invasion of an intact nonsister chromatid by only one of the two single-stranded tails (13). Single-end invasion (SEI) results in hybrid DNA (hDNA), in which the two strands in a duplex are of different parental origin. If the two parental duplexes are genetically different within the region of strand exchange, the resulting hDNA contains mismatched base pairs...

Formation of interference-sensitive meiotic cross-overs requires sufficient DNA leading-strand elongation

“…6E). This model is further supported by the finding that a weak mutation of yeast Pol-δ causes a reduction in the length of meiotic gene conversion tracts and CO number (15,36).…”

PCNA is efficiently loaded on the DNA recombination intermediate to modulate polymerase δ, η, and ζ activities

“…Pol δ is known as a replicative polymerase that constitutes the replisome (17,18). Genetic studies in yeast indicate that Pol δ is involved in mitotic gene conversion (19), meiotic recombination (20), repair of γ-ray-induced DNA damage (21), and homothallic switching (HO) endonuclease-induced gene conversion (22). Studies of HR in vertebrate systems have shown that translesion polymerases Pol η and Pol ζ also contribute to HR.…”

“…Genetic and biochemical studies in yeast indicated that both PCNA and Pol δ play major roles in HR (19)(20)(21)(22). Studies in vertebrate systems showed that Pol η and Pol ζ are also involved in HR (23,26).…”

A Novel Function of CRL4Cdt2

“…In addition to its roles in DNA replication, Pol ␦ also acts as the gap-filling enzyme in DNA repair processes. Evidence for this has been found in nucleotide excision repair (NER) (14,15), heteroduplex extension during homologous recombination, double strand break repair (16,17), and in translesion synthesis during the switch with translesion polymerases (18) in the DNA damage tolerance pathway (19,20). There is also evidence that Pol ␦ participates in long patch base excision repair (21).…”