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

Abstract

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.

REV1 promotes PCNA monoubiquitylation through interacting with ubiquitylated RAD18

“…In addition, although UV-induced PCNA-mUb was remarkably increased in XPRO30-Polη cells after adding doxycycline (Dox) to induce Polη expression ( Fig. 2A, lanes 1 and 3), which is in line with the previous report (Durando et al, 2013), overexpression of REV1 in the Polη-expressing XPRO30-Polη cells further increased the level of PCNA-mUb after UV damage ( Fig. 2A, lanes 3 and 4).…”

“…Given its key role in TLS and genome mutagenesis (Hoege et al, 2002;Moldovan et al, 2007;Chen et al, 2011), multiple studies have been performed to elucidate how the monoubiquitylation of PCNA is regulated in vivo (Hedglin and Benkovic, 2015). So far, a number of factors have been identified which regulate PCNA-mUb, including the RAD6-RAD18 ubiquitin ligase complex (Hoege et al, 2002;Kannouche et al, 2004), USP1 (Huang et al, 2006) and Polη (Durando et al, 2013). In this study, we report that REV1 also modulates PCNA-mUb in the absence of DNA damage, after exposure to UVC radiation, or treatment with hydroxyurea and MMC.…”

“…Previous studies have shown that REV1 interacts with Polη (Guo et al, 2003) and Polη regulates PCNA-mUb (Durando et al, 2013), leading us to wonder whether the stimulatory effect of REV1 on PCNA-mUb is mediated by Polη. We transfected GFP-REV1 into XP30RO-Polη cells and examined the level of PCNA-mUb in the presence and absence of Polη, and found that overexpression of REV1 still promoted PCNA-mUb in the absence of Polη (Fig.…”

Stability of the human polymerase δ holoenzyme and its implications in lagging strand DNA synthesis

“…4. In vivo, this full-length, human pol η mutant retains all biological functions except DNA synthesis activity (51,52). Thus, equivalent k off values for pol δ holoenzymes assembled with either PCNA or (Ub) 3 -PCNA suggests that the binding of TLS pols to PCNA is governed by the passive dissociation of pol δ.…”

Papel das proteínas XPD e DNA polimerase eta nas respostas de células humanas a danos no genoma

“…Células deficientes em pol  também mostraram-se mais sensíveis ao tratamento com doxorrubicina, assim como células deficientes em XP-A, sensibilidade que também é aumentada na presença de inibidores de quinases (Moraes et al, 2012b Rad18 para os locais onde há danos, o que é essencial para a monoubiquitinação de PCNA e consequentemente para a retirada do bloqueio da forquilha (Durando et al, 2013). …”