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Regulation of oxidative DNA damage repair by DNA polymerase ? and MutYH by cross-talk of phosphorylation and ubiquitination.

ABSTRACT: It is of pivotal importance for genome stability that repair DNA polymerases (Pols), such as Pols ? and ?, which all exhibit considerably reduced fidelity when replicating undamaged DNA, are tightly regulated, because their misregulation could lead to mutagenesis. Recently, we found that the correct repair of the abundant and highly miscoding oxidative DNA lesion 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-G) is performed by an accurate repair pathway that is coordinated by the MutY glycosylase homologue (MutYH) and Pol ? in vitro and in vivo. Pol ? is phosphorylated by Cdk2/cyclinA in late S and G2 phases of the cell cycle, promoting Pol ? stability by preventing it from being targeted for proteasomal degradation by ubiquitination. However, it has remained a mystery how the levels of Pol ? are controlled, how phosphorylation promotes its stability, and how the engagement of Pol ? in active repair complexes is coordinated. Here, we show that the E3 ligase Mule mediates the degradation of Pol ? and that the control of Pol ? levels by Mule has functional consequences for the ability of mammalian cells to deal with 8-oxo-G lesions. Furthermore, we demonstrate that phosphorylation of Pol ? by Cdk2/cyclinA counteracts its Mule-mediated degradation by promoting recruitment of Pol ? to chromatin into active 8-oxo-G repair complexes through an increase in Pol ?'s affinity to chromatin-bound MutYH. Finally, MutYH appears to promote the stability of Pol ? by binding it to chromatin. In contrast, Pol ? not engaged in active repair on chromatin is subject for proteasomal degradation.

SUBMITTER: Markkanen E

PROVIDER: S-EPMC3258598 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Regulation of oxidative DNA damage repair by DNA polymerase λ and MutYH by cross-talk of phosphorylation and ubiquitination.

Markkanen Enni E van Loon Barbara B Ferrari Elena E Parsons Jason L JL Dianov Grigory L GL Hübscher Ulrich U

Proceedings of the National Academy of Sciences of the United States of America 20111227 2

It is of pivotal importance for genome stability that repair DNA polymerases (Pols), such as Pols λ and β, which all exhibit considerably reduced fidelity when replicating undamaged DNA, are tightly regulated, because their misregulation could lead to mutagenesis. Recently, we found that the correct repair of the abundant and highly miscoding oxidative DNA lesion 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-G) is performed by an accurate repair pathway that is coordinated by the MutY glycosylase hom ...[more]

PMID: 22203964

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Project description:Recent reports have made important revelations, uncovering direct regulation of DNA damage response (DDR)-associated proteins and chromatin ubiquitination (Ubn) by macroautophagy/autophagy. Here, we report a previously unexplored connection between autophagy and DDR, via a deubiquitnase (DUB), USP14. Loss of autophagy in prostate cancer cells led to unrepaired DNA double-strand breaks (DSBs) as indicated by persistent ionizing radiation (IR)-induced foci (IRIF) formation for γH2AFX, and decreased protein levels and IRIF formation for RNF168, an E3-ubiquitin ligase essential for chromatin Ubn and recruitment of critical DDR effector proteins in response to DSBs, including TP53BP1. Consistently, RNF168-associated Ubn signaling and TP53BP1 IRIF formation were reduced in autophagy-deficient cells. An activity assay identified several DUBs, including USP14, which showed higher activity in autophagy-deficient cells. Importantly, inhibiting USP14 could overcome DDR defects in autophagy-deficient cells. USP14 IRIF formation and protein stability were increased in autophagy-deficient cells. Co-immunoprecipitation and colocalization of USP14 with MAP1LC3B and the UBA-domain of SQSTM1 identified USP14 as a substrate of autophagy and SQSTM1. Additionally, USP14 directly interacted with RNF168, which depended on the MIU1 domain of RNF168. These findings identify USP14 as a novel substrate of autophagy and regulation of RNF168-dependent Ubn and TP53BP1 recruitment by USP14 as a critical link between DDR and autophagy. Given the role of Ubn signaling in non-homologous end joining (NHEJ), the major pathway for repair of IR-induced DNA damage, these findings provide unique insights into the link between autophagy, DDR-associated Ubn signaling and NHEJ DNA repair.AbbreviationsATG7: autophagy related 7; CQ: chloroquine; DDR: DNA damage response; DUB: deubiquitinase; HR: homologous recombination; IR: ionizing radiation; IRIF: ionizing radiation-induced foci; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MIU1: motif interacting with ubiquitin; NHEJ: non homologous end-joining; PCa: prostate cancer; TP53BP1/53BP1: tumor protein p53 binding protein 1; RNF168: ring finger protein 168; SQSTM1/p62 sequestosome 1; γH2AFX/γH2AX: H2A histone family member X: phosphorylated, UBA: ubiquitin-associated; Ub: ubiquitin; Ubn: ubiquitination; USP14: ubiquitin specific peptidase 14.

Dataset Information

Regulation of oxidative DNA damage repair by DNA polymerase ? and MutYH by cross-talk of phosphorylation and ubiquitination.

Publications

Regulation of oxidative DNA damage repair by DNA polymerase λ and MutYH by cross-talk of phosphorylation and ubiquitination.

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