Abstract
DNA polymerase zeta (Pol zeta), a heterodimer of Rev3 and Rev7, is essential for DNA damage provoked mutagenesis in eukaryotes. DNA polymerases that function in a processive complex with the replication clamp proliferating cell nuclear antigen (PCNA) have been shown to possess a close match to the consensus PCNA-binding motif QxxLxxFF. This consensus motif is lacking in either subunit of Pol zeta, yet its activity is stimulated by PCNA. In particular, translesion synthesis of UV damage-containing DNA is dramatically stimulated by PCNA such that translesion synthesis rates are comparable with replication rates by Pol zeta on undamaged DNA. PCNA also stimulated translesion synthesis of a model abasic site by Pol zeta. Efficient PCNA stimulation required that PCNA was prevented from sliding off the damage-containing model oligonucleotide template-primer through the use of biotin-streptavidin bumpers or other blocks. Under those experimental conditions, facile bypass of the abasic site was also detected by DNA polymerase delta or eta (Rad30). The yeast DNA damage checkpoint clamp, consisting of Rad17, Mec3, and Ddc1, and an ortholog of human 9-1-1, has been implicated in damage-induced mutagenesis. However, this checkpoint clamp did not stimulate translesion synthesis by Pol zeta or by DNA polymerase delta.
Highlights
During the S phase of the eukaryotic cell cycle, when DNA damage may form a block for the high fidelity DNA polymerases such as DNA polymerase ␦ (Pol ␦)1 or Pol ⑀, several complex pathways are activated
A model has evolved in which Pol ␦ inserts a nucleotide across template damage, whereas Pol and Rev1 are responsible for extension of that inserted nucleotide
While studying the properties of Pol during DNA damage bypass synthesis, we found, surprisingly, that the synthetic efficiency of Pol is profoundly stimulated by proliferating cell nuclear antigen (PCNA) when the template DNA contains damage
Summary
During the S phase of the eukaryotic cell cycle, when DNA damage may form a block for the high fidelity DNA polymerases such as DNA polymerase ␦ (Pol ␦) or Pol ⑀, several complex pathways are activated. Most other types of damage are bypassed by a set of error-prone DNA polymerases, and this bypass forms the molecular basis for damage-induced mutagenesis in the cell. One of these translesion polymerases is Pol , a heterodimer of the Rev and Rev subunits, and the second enzyme is the Rev deoxycytidyl transferase [4, 5]. In analogy to bacterial mutagenesis systems, this model has been named the twopolymerase model for TLS [9, 10] This model has mainly been studied by measuring bypass rates and efficiencies of the catalytic cores of these enzymes without the inclusion of processivity factors such as the proliferating cell nuclear antigen (PCNA) [11, 12]. Our conclusion is that Pol interacts with PCNA via an as yet unidentified motif
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