Regulation of DNA repair by parkin
Introduction
Inherited mutations in parkin are a cause of autosomal recessive-juvenile Parkinsonism (AR-JP) [1]. Parkin contains a ubiquitin-like domain and two RING finger motifs, and functions as an E3 ubiquitin protein ligase [1]. A number of parkin substrates have been identified that are polyubiquitinated leading to their degradation by the proteasome [2], including CDC rel-1 [3], glycosylated α-synuclein [4], synphilin-1 [5], PAEL-R [6], cyclin E [7], aminoacyl-tRNA synthetase cofactor p38 [8], [9], and the PDZ domain-containing protein CASK/Lin2 [10]. In addition, parkin monoubiquitinates Eps15, an endocytic vesicle adaptor protein, and affects EGF receptor endocytosis and signaling [11].
A variety of parkin mutations, including point mutations, small deletions, and exonic duplications and deletions have been identified in families with inherited Parkinson’s disease (PD) [12]. Furthermore, the parkin gene is located in an unstable region of chromosome 6 that is frequently deleted in tumors. Point mutations, deletions, and exonic duplications have been identified in parkin in breast, lung, ovarian and hepatocellular carcinomas [13], [14], [15], [16]. Introduction of parkin into tumor cell lines reduced their proliferation [16], and ectopic parkin expression reduced tumorigenicity in nude mice [15], suggesting that parkin might function as a tumor suppressor.
Human genetic disorders of DNA repair are often associated with both neurodegeneration and increased cancer susceptibility. For example, loss of function mutations of the DNA damage response activator ATM in ataxia telangiectasia results in cerebellar Purkinje cell degeneration and increased susceptibility to cancer. Moreover, ATM-deficient mice exhibit selective degeneration of dopaminergic neurons of the substantia nigra [17], a neuronal population that also degenerates in PD. Hence, nigral dopaminergic neurons may be highly vulnerable to DNA damage, consistent with increased exposure to oxidative stress from dopamine metabolism. In this report, we show that parkin is required for optimal repair of DNA damage induced by UV-irradiation, and protects against genotoxicity. Moreover, parkin interacts with the proliferating cell nuclear antigen (PCNA), a protein that plays a central role in DNA excision repair.
Section snippets
Material and methods
Antibodies. Rabbit anti-parkin and mouse anti-Myc antibodies were from Cell Signaling. Mouse anti-PCNA antibody was from Santa Cruz Biotechnology.
Cell culture. SH-SY5Y and HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum. Wild-type and parkin −/− mouse ES cells were provided by Matthew Goldberg and Jie Shen [18]. Stable cell lines expressing wild-type parkin and the (Del 3–4) parkin mutant were established by transfecting pcDNA3.1(−)-parkin or pcDNA3.1(−)-parkin-Del
Parkin promotes DNA repair of UV-induced DNA damage
Parkin is a ubiquitin ligase and a candidate tumor suppressor. Both ubiquitin proteasome system and tumor suppressors play important roles in DNA damage response. We first asked whether endogenous parkin plays a role in DNA excision repair (NER), the primary pathway for repair of DNA damage induced by UV-irradiation. To quantify NER, we used a host cell reactivation assay that measures the activation of a luciferase reporter following UV-irradiation of the plasmid DNA in vitro[19], [21]. A
Discussion
These experiments indicate that parkin promotes DNA damage repair and protects against genotoxicity. Parkin-deficient cells show reduced DNA damage repair that can be restored by transfection of parkin. A parkin mutation associated with autosomal recessive-juvenile PD markedly reduces the DNA repair-promoting activity of parkin and increases vulnerability to DNA damage-induced cell death. These findings suggest a potential role for DNA damage in the pathogenesis of PD associated with parkin
Acknowledgments
I thank Matthew Goldberg and Jie Shen for providing parkin-deficient ES cells. I also thank Drs. Yihru Fannjiang, Matthew Hass and Yahua Chen for valuable discussion, and Monlan Yuan for assistance.
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