A non-catalytic role of DNA polymerase η in recruiting Rad18 and promoting PCNA monoubiquitination at stalled replication forks

Nucleic Acids Research, Mar 2013

Trans-lesion DNA synthesis (TLS) is a DNA damage-tolerance mechanism that uses low-fidelity DNA polymerases to replicate damaged DNA. The inherited cancer-propensity syndrome xeroderma pigmentosum variant (XPV) results from error-prone TLS of UV-damaged DNA. TLS is initiated when the Rad6/Rad18 complex monoubiquitinates proliferating cell nuclear antigen (PCNA), but the basis for recruitment of Rad18 to PCNA is not completely understood. Here, we show that Rad18 is targeted to PCNA by DNA polymerase eta (Polη), the XPV gene product that is mutated in XPV patients. The C-terminal domain of Polη binds to both Rad18 and PCNA and promotes PCNA monoubiquitination, a function unique to Polη among Y-family TLS polymerases and dissociable from its catalytic activity. Importantly, XPV cells expressing full-length catalytically-inactive Polη exhibit increased recruitment of other error-prone TLS polymerases (Polκ and Polι) after UV irradiation. These results define a novel non-catalytic role for Polη in promoting PCNA monoubiquitination and provide a new potential mechanism for mutagenesis and genome instability in XPV individuals.

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A non-catalytic role of DNA polymerase η in recruiting Rad18 and promoting PCNA monoubiquitination at stalled replication forks

Michael Durando 1 Satoshi Tateishi 0 Cyrus Vaziri 1 0 Division of Cell Maintenance, Institute of Molecular Embryology and Genetics (IMEG), Kumamoto University , Honjo 2-2-1, Kumamoto 860-0811, Japan 1 Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599, USA - Trans-lesion DNA synthesis (TLS) is a DNA damage-tolerance mechanism that uses low-fidelity DNA polymerases to replicate damaged DNA. The inherited cancer-propensity syndrome xeroderma pigmentosum variant (XPV) results from error-prone TLS of UV-damaged DNA. TLS is initiated when the Rad6/ Rad18 complex monoubiquitinates proliferating cell nuclear antigen (PCNA), but the basis for recruitment of Rad18 to PCNA is not completely understood. Here, we show that Rad18 is targeted to PCNA by DNA polymerase eta (Polg), the XPV gene product that is mutated in XPV patients. The C-terminal domain of Polg binds to both Rad18 and PCNA and promotes PCNA monoubiquitination, a function unique to Polg among Y-family TLS polymerases and dissociable from its catalytic activity. Importantly, XPV cells expressing full-length catalytically-inactive Polg exhibit increased recruitment of other error-prone TLS polymerases (Poli and Poli) after UV irradiation. These results define a novel non-catalytic role for Polg in promoting PCNA monoubiquitination and provide a new potential mechanism for mutagenesis and genome instability in XPV individuals. Living organisms are constantly exposed to ubiquitous genotoxins from endogenous and external sources (1). However, cells have evolved numerous DNA damage response (DDR) pathways that protect genomic DNA and prevent genetic instability (2). Trans-lesion synthesis (TLS) is a DDR mechanism involving specialized DNA polymerases that can replicate damaged DNA templates (3). TLS relies on inherently error-prone DNA polymerases of the Y family to replicate damaged DNA (4). TLS by Y-family polymerases (PolZ, Poli, Polk and Rev1) (5) maintains replication in cells harbouring damaged DNA, albeit at the cost of reduced fidelity. Each TLS polymerase performs relatively error-free replication past a preferred cognate lesion; in the absence of the appropriate TLS polymerase for its preferred lesion, mutagenic replication by error-prone polymerases predisposes to genetic instability (2). PolZ is unique among Y-family polymerases in its ability to perform accurate replication past UV-damaged DNA (6,7). Lack of PolZ in the inherited cancerpropensity syndrome xeroderma pigmentosum variant (XPV) (8) results in error-prone replication by other Y-family polymerases in sunlight-exposed cells (9,10). Thus, UV-induced mutagenesis due to PolZ deficiency compromises genetic integrity to manifest as exquisite sunlight sensitivity and early skin cancer propensity. A prerequisite for error-prone replication in TLS is the Rad6/Rad18-mediated monoubiquitination of proliferating cell nuclear antigen (PCNA) at the highly conserved lysine K164 (11,12). Y-family polymerases contain ubiquitin-binding (UBZ) domains that confer affinity to monoubiquitinated PCNA (13,14). Failure to monoubiquitinate PCNA at K164 phenocopies XPV by compromising TLS and sensitizing cells to UV light and other ubiquitous genotoxins (1518). Several other DDR pathways also depend on PCNA monoubiquitination, including SHPRH/HTLF-mediated template switching (19), ZRANB3-dependent replication fork restart (20), SNM1A-dependent intrastrand cross-link repair (21) and the Fanconi Anaemia pathway activation (22). Despite its pivotal role in the DDR, the molecular mechanisms regulating Rad18-mediated PCNA monoubiquitination are incompletely understood. The Rad18 Rad6 complex is thought to be recruited to the vicinity of damaged DNA via direct interactions with RPA-coated ssDNA (23,24). However, Rad18 lacks PCNA-binding motifs, and it is unclear how Rad18 is targeted specifically to PCNA at stalled forks (or other sites of post-replication repair). A recent report by Zou and colleagues (25) identified Spartan as a binding partner of both Rad18 and PCNA and proposed that Spartan acts as a scaffold for recruiting Rad18 to PCNA. Consistent with a role for Spartan in targeting Rad18 to PCNA, those workers found DNA damage-induced PCNA monoubiquitination was modestly attenuated in Spartan-depleted cells. However, several other more recent publications have reported alternative roles for Spartan in DNA damage signalling (2629), and it is unclear whether Spartan or alternative putative mediators exist to facilitate recruitment of Rad18 to PCNA. In mammalian cells, Rad18 exists in complex with PolZ (30,31), and association of Rad18 with PolZ is necessary for normal DNA damage tolerance (3032). Assembly of the Rad18PolZ complex is stringently controlled by Cdc7 and Chk1 kinases, which serve to integrate TLS with S-phase progression and the S-phase checkpoint, respectively (30,32). Here we report that the PolZRad18 interaction plays a (...truncated)


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Michael Durando, Satoshi Tateishi, Cyrus Vaziri. A non-catalytic role of DNA polymerase η in recruiting Rad18 and promoting PCNA monoubiquitination at stalled replication forks, Nucleic Acids Research, 2013, pp. 3079-3093, 41/5, DOI: 10.1093/nar/gkt016