Functional genomic screens identify CINP as a genome maintenance protein

Lovejoy, Courtney A.; Xu, Xin; Bansbach, Carol E.; Glick, Gloria G.; Zhao, Runxiang; Ye, Fei; Sirbu, Bianca M.; Titus, Laura C.; Shyr, Yu; Chen, David

doi:10.1073/pnas.0909345106

Cited by 62 publications

(68 citation statements)

References 44 publications

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“…pEGFP was purchased from Clontech. pEGFPTopBP1-978-1286 wild type (also referred to as GFP-TopBP1-AAD below), pEGFP-TopBP1-978-1286 W1145R, Myc-TopBP1, and HA-CINP were provided by David Cortez (Vanderbilt University School of Medicine, Nashville, TN) (28,29). Flag-Hus1 and Flag-Rad17 were purchased from Origene.…”

Section: Methodsmentioning

confidence: 99%

Efficient Herpes Simplex Virus 1 Replication Requires Cellular ATR Pathway Proteins

Mohni

Dee

Smith

et al. 2013

J Virol

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aHerpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that replicates in the nucleus of the host cell and is known to interact with several components of the cellular DNA-damage-signaling machinery. We have previously reported that the DNA damage response kinase, ATR, is specifically inactivated in HSV-1-infected cells. On the other hand, we have also shown that ATR and its scaffolding protein, ATRIP, are recruited to viral replication compartments, where they play beneficial roles during HSV-1 replication. In order to better understand this apparent discrepancy, we tested the hypothesis that some of the components of the ATR pathway may exert an antiviral effect on infection. In fact, we learned that all 10 of the canonical ATR pathway proteins are stable in HSV-infected cells and are recruited to viral replication compartments; furthermore, short hairpin RNA (shRNA) knockdown shows that several, including ATRIP, RPA70, TopBP1, Claspin, and CINP, are required for efficient HSV-1 replication. We also determined that activation of the ATR kinase prior to infection did not affect virus yield but did result in reduced levels of recombination between coinfecting viruses. Together, these data suggest that ATR pathway proteins are not antiviral per se but that activation of ATR signaling may have negative consequences during viral replication, such as inhibiting recombination.

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Section: Methodsmentioning

confidence: 99%

Efficient Herpes Simplex Virus 1 Replication Requires Cellular ATR Pathway Proteins

Mohni

Dee

Smith

et al. 2013

J Virol

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“…Identifies USP11-We previously used activation of the DNA damage response as a reporter to identify genome maintenance activities in mammalian cells (44). 73 genes were identified that caused increased DDR signaling when silenced by RNAi even in the absence of any added genotoxic agent.…”

Section: Rnai Screen For Parp Inhibitor (Parpi) Hypersensitivitymentioning

confidence: 99%

Sensitivity to Poly(ADP-ribose) Polymerase (PARP) Inhibition Identifies Ubiquitin-specific Peptidase 11 (USP11) as a Regulator of DNA Double-strand Break Repair

Wiltshire

Lovejoy

Wang

et al. 2010

Journal of Biological Chemistry

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118

116

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DNA damage repair and checkpoint responses prevent genome instability and provide a barrier to the development of cancer. Inherited mutations in DNA damage response (DDR) genes such as those that encode the homologous recombination (HR) proteins BRCA1 and BRCA2 cause cancer predisposition syndromes. PARP inhibitors are an exciting new class of targeted therapy for treating patients with HR repair-defective tumors. In this study, we use an RNAi screen to identify genes that when silenced cause synthetic lethality with the PARP inhibitor AZD2281. This screen identified the deubiquitylating enzyme USP11 as a participant in HR repair of DNA doublestrand breaks. Silencing USP11 with siRNA leads to spontaneous DDR activation in otherwise undamaged cells and hypersensitivity to PARP inhibition, ionizing radiation, and other genotoxic stress agents. Moreover, we demonstrate that HR repair is defective in USP11-silenced cells. Finally, the recruitment of a subset of double-strand break repair proteins including RAD51 and 53BP1 to repair foci is misregulated in the absence of USP11 catalytic activity. Thus, our synthetic lethal approach identified USP11 as a component of the HR doublestrand break repair pathway.Faithful DNA replication and repair is critical to maintain genome integrity. An intact DNA damage response (DDR) 2 prevents mutations and gross chromosomal instability. Defects in the DDR cause cancer predisposition syndromes, demonstrating the importance of the DDR as a barrier to tumorigenesis (1, 2). Most cancers have defects in some part of the DDR. This difference between normal and cancer cells not only explains the genetic instability associated with cancer, but also provides an opportunity for therapeutic intervention. This concept is the basis for many genotoxic therapies where the DNA damage is repaired in healthy cells but has lethal consequences in cancer cells.Targeted therapy of tumors is a method of treatment that seeks to maximize response while reducing harmful side effects to healthy cells. Inhibition of the DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP1) is a recently developed strategy for cancer therapy that exploits DDR defects in a subset of cancers (3). PARP1 is a DNA repair enzyme that is responsible for the sensing and repair of single-strand DNA breaks via short-patch base-excision repair (4). Thousands of single-strand breaks occur per cell per day. When a replication fork encounters a single-strand break, the result is collapse of the fork and formation of a double-strand break (5). In wildtype cells, these replication-associated double-strand breaks are often repaired via homologous recombination (HR). Cells deficient in PARP activity have increased recombination as evidenced by elevated rates of sister chromatid exchange (6) and increased intranuclear foci of the RAD51 recombination protein (7,8). Cells deficient in HR proteins such as BRCA1 and BRCA2 are unable to repair these double-strand breaks efficiently and die (7,8). Thus, PARP inhibitors are being successfully used t...

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“…Weak CINP sumoylation has been observed by another group through a screening system, suggesting the existence of other modified types of CINP (44). Interestingly, Lovejoy et al (38) and another group (45) recently identified CINP as a checkpoint protein to maintain genomic stability, thus shedding light on the function of CINP in genomic maintenance and cell cycle regulation.…”

Section: Discussionmentioning

confidence: 99%

“…Diminished Rb expression can also lead to inappropriate DNA synthesis following a senescence trigger (37). Of note, CINP has recently been identified as a genome maintenance protein (38), and it is known that the sensing and repairing of DNA damage are involved in cell cycle progression. Therefore, we theorized that the DNA damage response could be exploited by NS5B to delay cells in S phase.…”

Section: Ns5b/cinp Association Is Responsible For S Phase Delay and Smentioning

confidence: 99%

Hepatitis C Virus NS5B Protein Delays S Phase Progression in Human Hepatocyte-derived Cells by Relocalizing Cyclin-dependent Kinase 2-interacting Protein (CINP)

Wang

et al. 2011

Journal of Biological Chemistry

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Cell cycle dysregulation is a critical event in virus infectionassociated tumorigenesis. Previous studies have suggested that hepatitis C virus NS5B modulates cell cycle progression in addition to participating in RNA synthesis as an RNA-dependent RNA polymerase. However, the molecular mechanisms have thus far remained unclear. In this study, a HepG2 Tet-On NS5B stable cell line was generated to confirm the effect of NS5B on the cell cycle. To better understand the role of NS5B in cell cycle regulation, yeast two-hybrid assays were performed using a human liver cDNA library. The cyclin-dependent kinase 2-interacting protein (CINP) was identified. The interaction between NS5B and CINP was further demonstrated by in vivo and in vitro assays, and their association was found to be indispensable for S phase delay and cell proliferation suppression. Further experiments indicated that NS5B relocalized CINP from the nucleus to the cytoplasm. Directly knocking down CINP by specific siRNA resulted in a significant alteration in the DNA damage response and expression of cell cycle checkpoint proteins, including an increase in p21 and a decrease in phosphorylated Retinoblastoma and Chk1. Similar results were observed in cells expressing NS5B, and the effects were partially reversed upon ectopic overexpression of CINP. These studies suggest that the DNA damage response might be exploited by NS5B to hinder cell cycle progression. Taken together, our data demonstrate that NS5B delays cells in S phase through interaction with CINP and relocalization of the protein from the nucleus to the cytoplasm. Such effects might contribute to hepatitis C virus persistence and pathogenesis.

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Functional genomic screens identify CINP as a genome maintenance protein

Cited by 62 publications

References 44 publications

Efficient Herpes Simplex Virus 1 Replication Requires Cellular ATR Pathway Proteins

Efficient Herpes Simplex Virus 1 Replication Requires Cellular ATR Pathway Proteins

Sensitivity to Poly(ADP-ribose) Polymerase (PARP) Inhibition Identifies Ubiquitin-specific Peptidase 11 (USP11) as a Regulator of DNA Double-strand Break Repair

Hepatitis C Virus NS5B Protein Delays S Phase Progression in Human Hepatocyte-derived Cells by Relocalizing Cyclin-dependent Kinase 2-interacting Protein (CINP)

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