PCNA is ubiquitinated by RNF8

Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.
Cell cycle (Georgetown, Tex.) (Impact Factor: 4.57). 12/2008; 7(21):3399-404. DOI: 10.4161/cc.7.21.6949
Source: PubMed


The ubiquitination of PCNA is an essential event in the operation of the DNA Damage Tolerance (DDT) pathway that is activated after DNA damage caused by UV or chemical agents during S-phase. This pathway allows the bypass of DNA damage by translesion synthesis that would otherwise cause replication fork stalling. PCNA is mono-ubiquitinated by Rad18-Rad6, and polyubiquitinated by Rad5-Ubc13/Uev1 in the DDT pathway. Mono-and polyubiquitination of PCNA are key processes in the translesion bypass and template switching sub-pathways of the DDT. DNA damage by IR causes DSBs, which trigger the DNA Damage Response (DDR). The ubiquitin ligase RNF8 has a critical role in the assembly of BRCA1 complexes at the DSBs in the DDR. We show that RNF8 readily mono-ubiquitinates PCNA in the presence of UbcH5c, and polyubiquitinates PCNA in the added presence of Ubc13/Uev1a. These reactions are the same as those performed by Rad18-Rad6 and Rad5-Ubc13. RNF8 depletion suppressed both UV and MNNG-stimulated mono-ubiquitination of PCNA, revealing that an RNF8-dependent pathway for PCNA ubiquitination is operative in vivo. These findings provide evidence that RNF8, a key E3 ligase in the DDR, may also play a role in the DDT.

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Available from: Sufang Zhang, Feb 06, 2014
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    • "Inaddition,RNF8alsoubiquitylatesK48-dependentsub- stratessuchasthelysinedemethylaseJMJD2A(Malletteetal., 2012),theNHEJrepairproteinKu80(FengandChen,2012), andtheDNApolymeraseslidingclampproliferatingcellnuclear antigen(PCNA),whichisinvolvedinDNAsynthesisandrepair (Zhangetal.,2008).Consequently,theseproteinsareremoved fromchromatinforproteasomaldegradation. "
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    ABSTRACT: In response to DNA damage, cells activate a highly conserved and complex kinase-based signaling network, commonly referred to as the DNA damage response (DDR), to safeguard genomic integrity. The DDR consists of a set of tightly regulated events, including detection of DNA damage, accumulation of DNA repair factors at the site of damage, and finally physical repair of the lesion. Upon overwhelming damage the DDR provokes detrimental cellular actions by involving the apoptotic machinery and inducing a coordinated demise of the damaged cells (DNA damage-induced apoptosis, DDIA). These diverse actions involve transcriptional activation of several genes that govern the DDR. Moreover, recent observations highlighted the role of ubiquitylation in orchestrating the DDR, providing a dynamic cellular regulatory circuit helping to guarantee genomic stability and cellular homeostasis (Popovic et al., 2014). One of the hallmarks of human cancer is genomic instability (Hanahan and Weinberg, 2011). Not surprisingly, deregulation of the DDR can lead to human diseases, including cancer, and can induce resistance to genotoxic anti-cancer therapy (Lord and Ashworth, 2012). Here, we summarize the role of ubiquitin-signaling in the DDR with special emphasis on its role in cancer and highlight the therapeutic value of the ubiquitin-conjugation machinery as a target in anti-cancer treatment strategy.
    Frontiers in Genetics 04/2015; 1(6). DOI:10.3389/fgene.2015.00098
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    • "Following replication arrest, eukaryotic PCNA is monoubiquitinated at lysine 164 in a reaction mediated by the E3 ubiquitin ligase RAD18 working in conjunction with the E2 ubiquitinconjugating enzyme RAD6 (Hoege et al. 2002; Stelter and Ulrich 2003; Haracska et al. 2004; Kannouche et al. 2004). In vertebrates, there are additional ubiquitin ligases capable of introducing this modification (Simpson et al. 2006; Zhang et al. 2008; Terai et al. 2010). The activity of RAD18 is stimulated by its association with RPA-coated single-stranded DNA (Davies et al. 2008), and PCNA monoubiquitation serves to recruit the Y-family polymerases to sites of replication arrest by enhancing their direct association with PCNA through an interaction between ubiquitin-PCNA and ubiquitin-binding motif (UBM) or ubiquitin-binding zinc (UBZ) domains in the carboxyl terminus of the polymerases (Bienko et al. 2005). "
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    ABSTRACT: The structural features that enable replicative DNA polymerases to synthesize DNA rapidly and accurately also limit their ability to copy damaged DNA. Direct replication of DNA damage is termed translesion synthesis (TLS), a mechanism conserved from bacteria to mammals and executed by an array of specialized DNA polymerases. This chapter examines how these translesion polymerases replicate damaged DNA and how they are regulated to balance their ability to replicate DNA lesions with the risk of undesirable mutagenesis. It also discusses how TLS is co-opted to increase the diversity of the immunoglobulin gene hypermutation and the contribution it makes to the mutations that sculpt the genome of cancer cells.
    Cold Spring Harbor perspectives in biology 03/2013; 5(3). DOI:10.1101/cshperspect.a012708 · 8.68 Impact Factor
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    • "This presumption is supported by a number of examples of E3 ligases involved in the DDRs [Kannouche et al., 2004; Harper and Elledge, 2007; Yan and Jetten, 2008; Hannah and Zhou, 2009], whose actions are dependent on their appropriate recruitment to DNA-damage sites, for example, monoubiquitination of PCNA is mediated by Rad18/Rad6 [Hoege et al., 2002] and takes place in situ on DNA [Kannouche et al., 2004]. In addition, RNF8, an E3 ligase, which plays a key role in assembly of the signaling complexes in DDR, also ubiquitinates PCNA in vivo [Zhang et al., 2008]. Little is known as yet of the E3 ligases that are involved in p12 degradation. "
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    ABSTRACT: The p12 subunit of polymerase delta (Pol δ) is degraded in response to DNA damage induced by UV, alkylating agents, oxidative, and replication stresses. This leads to the conversion of the Pol δ4 holoenzyme to the heterotrimer, Pol δ3. We review studies that establish that Pol δ3 formation is an event that could have a major impact on cellular processes in genomic surveillance, DNA replication, and DNA repair. p12 degradation is dependent on the apical ataxia telangiectasia and Rad3 related (ATR) kinase and is mediated by the ubiquitin-proteasome system. Pol δ3 exhibits properties of an "antimutator" polymerase, suggesting that it could contribute to an increased surveillance against mutagenesis, for example, when Pol δ carries out bypass synthesis past small base lesions that engage in spurious base pairing. Chromatin immunoprecipitation analysis and examination of the spatiotemporal recruitment of Pol δ to sites of DNA damage show that Pol δ3 is the primary form of Pol δ associated with cyclobutane pyrimidine dimer lesions and therefore should be considered as the operative form of Pol δ engaged in DNA repair. We propose a model for the switching of Pol δ with translesion polymerases, incorporating the salient features of the recently determined structure of monoubiquitinated proliferating cell nuclear antigen and emphasizing the role of Pol δ3. Because of the critical role of Pol δ activity in DNA replication and repair, the formation of Pol δ3 in response to DNA damage opens the prospect that pleiotropic effects may ensue. This opens the horizons for future exploration of how this novel response to DNA damage contributes to genomic stability. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.
    Environmental and Molecular Mutagenesis 12/2012; 53(9). DOI:10.1002/em.21743 · 2.63 Impact Factor
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