Analysis of CPD ultraviolet lesion bypass in chicken DT40 cells: polymerase η and PCNA ubiquitylation play identical roles.

Translesion synthesis (TLS) provides a mechanism of copying damaged templates during DNA replication. This potentially mutagenic process may operate either at the replication fork or at post-replicative gaps. We used the example of T-T cyclobutane pyrimidine dimer (CPD) bypass to determine the influence of polymerase recruitment via PCNA ubiquitylation versus the REV1 protein on the efficiency and mutagenic outcome of TLS. Using mutant chicken DT40 cell lines we show that, on this numerically most important UV lesion, defects in polymerase η or in PCNA ubiquitylation similarly result in the long-term failure of lesion bypass with persistent strand gaps opposite the lesion, and the elevation of mutations amongst successful TLS events. Our data suggest that PCNA ubiquitylation promotes CPD bypass mainly by recruiting polymerase η, resulting in the majority of CPD lesions bypassed in an error-free manner. In contrast, we find that polymerase ζ is responsible for the majority of CPD-dependent mutations, but has no essential function in the completion of bypass. These findings point to a hierarchy of access of the different TLS polymerases to the lesion, suggesting a temporal order of their recruitment. The similarity of REV1 and REV3 mutant phenotypes confirms that the involvement of polymerase ζ in TLS is largely determined by its recruitment to DNA by REV1. Our data demonstrate the influence of the TLS polymerase recruitment mechanism on the success and accuracy of bypass.

Fluorescence detection of the endonuclease III reaction using modified oligonucleotides.

We have developed a method for the detection of the endonuclease III reaction by fluorescence. The probes were 13-base-pair hairpin-shaped oligonucleotides containing one of the isomers of thymine glycol or 5,6-dihydrothymine as a damaged base at the center, and had a fluorophore and a quencher at the 5' and 3' ends, respectively. Fluorescence was detected when the probe was cleaved by the enzyme, because the short fragment bearing the fluorophore could not be hybridized to the quencher strand at the incubation temperature. The substrate specificity was shown using Escherichia coli and human enzymes.

REV1 restrains DNA polymerase zeta to ensure frame fidelity during translesion synthesis of UV photoproducts in vivo.

Exposure to ultraviolet light induces a number of forms of damage in DNA, of which (6-4) photoproducts present the most formidable challenge to DNA replication. No single DNA polymerase has been shown to bypass these lesions efficiently in vitro suggesting that the coordinate use of a number of different enzymes is required in vivo. To further understand the mechanisms and control of lesion bypass in vivo, we have devised a plasmid-based system to study the replication of site-specific T-T(6-4) photoproducts in chicken DT40 cells. We show that DNA polymerase zeta is absolutely required for translesion synthesis (TLS) of this lesion, while loss of DNA polymerase eta has no detectable effect. We also show that either the polymerase-binding domain of REV1 or ubiquitinated PCNA is required for the recruitment of Polzeta as the catalytic TLS polymerase. Finally, we demonstrate a previously unappreciated role for REV1 in ensuring bypass synthesis remains in frame with the template. Our data therefore suggest that REV1 not only helps to coordinate the delivery of DNA polymerase zeta to a stalled primer terminus but also restrains its activity to ensure that nucleotides are incorporated in register with the template strand.