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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    • "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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    • "The priming activity of RNF168 and UbcH5c is required for chain formation, because the use of a ligase-inactive mutant (Brzovic et al., 2003) of RNF168 results in unmodified H2A (Figure 5C). Furthermore , the addition of RNF8 and Ubc13/Mms2 on other monoubiquitinated substrates (e.g., H2A ubiquitinated by Ring1B, PCNA (Zhang et al., 2008), or H2B) also leads to K63 chain formation on Figure 5. K63-Linked Ubiquitin Chains on H2A and H2AX Are Specifically Conjugated to K13-15 (A) When RNF8 is added to UbcH5c and RNF168, no additional activity is observed toward H2A (lane 5-6) over RNF168 activity. When Ubc13/Mms2 is added, RNF8 makes chains on ubiquitinated H2A (lane 11-12). "
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    ABSTRACT: Ubiquitin-dependent signaling during the DNA damage response (DDR) to double-strand breaks (DSBs) is initiated by two E3 ligases, RNF8 and RNF168, targeting histone H2A and H2AX. RNF8 is the first ligase recruited to the damage site, and RNF168 follows RNF8-dependent ubiquitination. This suggests that RNF8 initiates H2A/H2AX ubiquitination with K63-linked ubiquitin chains and RNF168 extends them. Here, we show that RNF8 is inactive toward nucleosomal H2A, whereas RNF168 catalyzes the monoubiquitination of the histones specifically on K13-15. Structure-based mutagenesis of RNF8 and RNF168 RING domains shows that a charged residue determines whether nucleosomal proteins are recognized. We find that K63 ubiquitin chains are conjugated to RNF168-dependent H2A/H2AX monoubiquitination at K13-15 and not on K118-119. Using a mutant of RNF168 unable to target histones but still catalyzing ubiquitin chains at DSBs, we show that ubiquitin chains per se are insufficient for signaling, but RNF168 target ubiquitination is required for DDR.
    Cell 09/2012; 150(6):1182-95. DOI:10.1016/j.cell.2012.08.005 · 32.24 Impact Factor
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