Article

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

ABSTRACT

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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    • "Concentrations of Pol δ (p125 subunit content), PDIP46 and its mutants were determined by SDS-PAGE with a range of concentrations of catalase as protein standard (Fig. S6, Supplementary Data). Human PCNA was expressed in E. coli and purified as previously described[81,82]. "
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    ABSTRACT: PDIP46 (SKAR, POLDIP3) was discovered through its interaction with the p50 subunit of human DNA polymerase δ (Pol δ). Its functions in DNA replication are unknown. PDIP46 associates with Pol δ in cell extracts both by immunochemical and protein separation methods, as well as by ChIP analyses. PDIP46 also interacts with PCNA via multiple copies of a novel PCNA binding motif, the APIMs (AlkB homologue-2 PCNA-Interacting Motif). Sites for both p50 and PCNA binding were mapped to the N-terminal region containing the APIMs. Functional assays for the effects of PDIP46 on Pol δ activity on singly primed ssM13 DNA templates revealed that it is a novel and potent activator of Pol δ. The effects of PDIP46 on Pol δ in primer extension, strand displacement and synthesis through simple hairpin structures reveal a mechanism where PDIP46 facilitates Pol δ4 synthesis through regions of secondary structure on complex templates. In addition, evidence was obtained that PDIP46 is also capable of exerting its effects by a direct interaction with Pol δ, independent of PCNA. Mutation of the Pol δ and PCNA binding region resulted in a loss of PDIP46 functions. These studies support the view that PDIP46 is a novel accessory protein for Pol δ that is involved in cellular DNA replication. This raises the possibility that altered expression of PDIP46 or its mutation may affect Pol δ functions in vivo, and thereby be a nexus for altered genomic stability.
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    • "Given that PCNA-mUb promotes TLS pathway (Hoege et al., 2002;Moldovan et al., 2007;Chen et al., 2011), many studies have been performed to understand how this modification happens in vivo (Hedglin and Benkovic, 2015). In addition to the major E3 ubiquitin ligase RAD18, several other E3 ligases, like RNF8 (Zhang et al., 2008) and CRL cdt2 (Terai et al., 2010), have been reported to regulate PCNA-mUb. Additionally, many factors, including SIVA1 (Han et al., 2014), Spartan/C1orf124 (protein with sprT-like domain at the N terminus, also known as DVC1 [DNA damage protein targeting VCP]) (Centore et al., 2012), MSH2 (MutS protein homolog 2) (Zlatanou et al., 2011;Lv et al., 2013), BRCA1 (breast cancer 1) (Tian et al., 2013), have also been found to regulate the RAD18-dependent PCNA-mUb. "
    Zhifeng Wang · Min Huang · Xiaolu Ma · Huiming Li · Tieshan Tang · Caixia Guo
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    ABSTRACT: Translesion DNA synthesis (TLS) is one mode of DNA damage tolerance, which plays an important role in genome mutagenesis and chromatin integrity maintenance. PCNA monoubiquitination is one of the key factors for TLS pathway choice. So far, it remains unclear how TLS pathway is elaborately regulated. Here, we report that TLS polymerase REV1 can promote PCNA monoubiquitination after UV radiation. Further studies revealed that this stimulatory effect is mediated through the enhanced interaction between REV1 and ubiquitinated RAD18, which facilitates the release of nonubiquitinated RAD18 from ubiquitinated RAD18 trapping followed by more RAD18 recruiting to chromatin for its TLS function. Furthermore, we found that this stimulatory effect could also be detected after exposure to hydroxyurea or mitomycin C, but not methyl methanesulfonate (MMS), which is in line with the fact that ubiquitinated RAD18 could not be detected after exposure to MMS.
    Full-text · Article · Jan 2016 · Journal of Cell Science
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    • "Concentrations of Pol δ (p125 subunit content), PDIP46 and its mutants were determined by SDS-PAGE with a range of concentrations of catalase as protein standard (Fig. S6, Supplementary Data). Human PCNA was expressed in E. coli and purified as previously described[81,82]. "
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    ABSTRACT: PDIP46 (poldip3) is a 46 kDa protein which interacts with human DNA polymerase δ (Pol δ) via the p50 subunit, and whose biochemical effects on Pol δ are unknown. The objectives of this study were to characterize the effects of PDIP46 on Pol δ activity. The interaction sites between the p50 subunit of Pol δ and PDIP46 were mapped. PDIP46 was also found to interact with PCNA, which it does via multiple copies of a novel PCNA binding motif (APIM - AlkB homologue 2 PCNA-Interacting Motif). Both the p50 and PCNA interaction regions are located in the N-terminus. PDIP46 potently activates Pol δ activity (ca. 10-fold with an apparent KD of ca. 34 nM) on singly primed 7 kb ssM13 DNA in an assay which requires highly processive synthesis. Further dissection of the effects of PDIP46 using model oligonucleotide templates showed that it stimulated primer extension and strand displacement reactions in the presence of PCNA. PDIP46 also stimulated primer extension in the absence of PCNA, indicating that it has the ability to exert a direct effect on Pol δ. PDIP46 was associated with Pol δ on DNA as shown by chromatin immunoprecipitation using antibody against the Pol δ p125 subunit. These in vitro studies show that PDIP46 has the biochemical attributes to function as a novel accessory protein for Pol δ that accelerates its activity, and thus is a candidate for a role as a cellular regulator of Pol δ.
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