The translesion DNA polymerases Pol ζ and Rev1 are activated independently of PCNA ubiquitination upon UV radiation in mutants of DNA polymerase δ.

Carine Tellier-Lebegue,Xavier Veaute,Eric Coïc,Emilie Ma,Laurent Maloisel,Eléa Dizet,Jean-Baptiste Charbonnier

doi:10.1371/journal.pgen.1007119

Abstract

Replicative DNA polymerases cannot insert efficiently nucleotides at sites of base lesions. This function is taken over by specialized translesion DNA synthesis (TLS) polymerases to allow DNA replication completion in the presence of DNA damage. In eukaryotes, Rad6- and Rad18-mediated PCNA ubiquitination at lysine 164 promotes recruitment of TLS polymerases, allowing cells to efficiently cope with DNA damage. However, several studies showed that TLS polymerases can be recruited also in the absence of PCNA ubiquitination. We hypothesized that the stability of the interactions between DNA polymerase δ (Pol δ) subunits and/or between Pol δ and PCNA at the primer/template junction is a crucial factor to determine the requirement of PCNA ubiquitination. To test this hypothesis, we used a structural mutant of Pol δ in which the interaction between Pol3 and Pol31 is inhibited. We found that in yeast, rad18Δ-associated UV hypersensitivity is suppressed by pol3-ct, a mutant allele of the POL3 gene that encodes the catalytic subunit of replicative Pol δ. pol3-ct suppressor effect was specifically dependent on the Rev1 and Pol ζ TLS polymerases. This result strongly suggests that TLS polymerases could rely much less on PCNA ubiquitination when Pol δ interaction with PCNA is partially compromised by mutations. In agreement with this model, we found that the pol3-FI allele suppressed rad18Δ-associated UV sensitivity as observed for pol3-ct. This POL3 allele carries mutations within a putative PCNA Interacting Peptide (PIP) motif. We then provided molecular and genetic evidence that this motif could contribute to Pol δ-PCNA interaction indirectly, although it is not a bona fide PIP. Overall, our results suggest that the primary role of PCNA ubiquitination is to allow TLS polymerases to outcompete Pol δ for PCNA access upon DNA damage.

Highlights

Despite the remarkable catalytic activities of replicative DNA polymerases, these enzymes cannot efficiently incorporate nucleotides opposite damaged template DNA
These translesion polymerases are not processive and they are poorly accurate. They need to be tightly regulated. This is achieved by the covalent binding of the small ubiquitin peptide to the polymerase cofactor PCNA that subsequently triggers the recruitment of translesion polymerases at sites of DNA damage
We present strong genetic evidence in Saccharomyces cerevisiae implying that the replicative DNA polymerase δ (Pol δ) prevents the recruitment of the translesion polymerases Pol z and Rev1 following UV irradiation unless PCNA is ubiquitinated

Summary

Introduction

Despite the remarkable catalytic activities of replicative DNA polymerases, these enzymes cannot efficiently incorporate nucleotides opposite damaged template DNA. The inevitable consequence of the TLS polymerases feature to synthesize across DNA lesions with no associated proofreading activity, is their overall reduced fidelity, even at undamaged templates. This could lead to accumulation of unwanted mutations and their activity needs to be tightly regulated [2,3]. The main mechanism of damage-induced activation of TLS polymerases involves covalent modifications of the sliding clamp PCNA by ubiquitin or SUMO peptides [4,5,6,7]. PCNA poly-ubiquitination triggers a template switch mechanism [4,10]

Methods

Findings

Discussion

Conclusion