Proliferating Cell Nuclear Antigen Promotes Translesion Synthesis by DNA Polymerase ζ

Abstract

DNA polymerase (Pol ), 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 , 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 on undamaged DNA. PCNA also stimulated translesion synthesis of a model abasic site by Pol . Efficient PCNA stimulation required that PCNA was prevented from sliding off the damagecontaining 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 ␦ or (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 damageinduced mutagenesis. However, this checkpoint clamp did not stimulate translesion synthesis by Pol or by DNA polymerase ␦.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. The Saccharomyces cerevisiae RAD6 and RAD18 genes control a bifurcating pathway that activates translesion synthesis (TLS) by error-prone DNA polymerases as well as a recombinational damage avoidance pathway (reviewed in Ref. 1). Pol is largely responsible for the relatively error-free bypass of cis-syn pyrimidine dimers in the template strand (2, 3). In contrast, 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 Rev3 and Rev7 subunits, and the second enzyme is the Rev1 deoxycytidyl transferase (4, 5). The replicative polymerase Pol ␦ has also been shown to be required for damage-induced mutagenesis, primarily through mutational studies of its Pol32 subunit (3, 6 -8). Consequently, 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. In analogy to bacterial mutagenesis systems, this model has been named the twopolymerase model for TLS (9, 10). Biochemically, 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).Most DNA polymerases possess a unique consensus motif, QxxLxxFF, that is required for binding of the polymerase to PCNA and for PCNA-mediated processive DNA synthesis (reviewed in (13)). For ...

Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases η and REV1

“…Most other types of damage are bypassed by the replicative Pol ␦ in combination with the error-prone Pol and the Rev1 deoxycytidyl transferase, and this bypass forms the molecular basis for damage-induced mutagenesis in the cell. Notably, three of these four enzymes, Pol ␦, Pol , and Pol , carry out both processive DNA synthesis and damage bypass with unmodified PCNA as sliding clamp (27,43). We determined the effect of PCNA ubiquitination on TLS by these three DNA polymerases and by Rev1.…”

“…These PCNA-stimulated error-prone polymerases include yeast and human Pol , and human Pol and Pol (23)(24)(25)(26). In addition, the mutagenic Pol is also stimulated by PCNA (27). Therefore, data indicating that a TLS polymerase functions specifically with PCNA Ubi remain unclear.…”

“…mp18 ssDNA primed with a single primer was used in the processivity assays (29). All oligonucleotide substrates were prepared as described (27). The linear oligonucleotide templates contained a biotin at both ends, to which a 2-fold molar excess of streptavidin was added.…”

DNA polymerase zeta (pol ζ) in higher eukaryotes

“…Embryonic stem cells harboring this mutation have an 23], via the Rev 7 subunit [23]. PCNA can stimulate DNA synthesis by pol ζ [22]. There is evidence that a Rev3-Rev7-Rev1 complex associates with monoubiquitinated-PCNA through ubiquitin-binding motifs in REV1, which helps to enable pol ζ or other REV1-interacting bypass polymerases to insert a base opposite damage and then extend from the resulting non-standard primer-template [22,24] (see the review by Andersen et al in this issue [25]).…”

DNA Replication to Aid Somatic Hypermutation

“…Monoubiquitinated PCNA Lys164 constitutes another binding motif that is recognized by the ubiquitin-binding domain (UBD) in pol η, pol ι (Bienko et al, 2005) and possibly other TLS polymerases. Further, PCNA, together with RFC and RPA, strongly enhances the insertion activity of human pol η (Haracska et al, 2001a;Haracska et al, 2001b) and PCNA binds to pol ζ and strongly stimulates its TLS activity to copy UV-damaged DNA template in vitro (Garg et al, 2005), suggesting that PCNA functions not only as an "adaptor" but also as an "inducer" of TLS polymerases during SHM. PCNA might also interact with pol θ and augment its abasic site bypassing, mismatch insertion and extension functions.…”