Molecular and Cellular Pathobiology Human Nuclease/Helicase DNA2 Alleviates Replication Stress by Promoting DNA End Resection
چکیده
In precancerous and cancerous lesions, excessive growth signals resulting from activation of oncogenes or loss of tumor suppressor genes lead to intensive replication stress, which is recognized by a high level of replicationassociated DNA double-strand breaks (DSB). However, the molecular mechanism by which cells alleviate excessive replication stress remains unclear. In this study, we report that the human nuclease/helicase DNA2 facilitates homologous recombination to repair replication-associated DNA DSBs, thereby providing cells with survival advantages under conditions of replication stress. The nuclease activity of DNA2 was required for DSB end resection, which allowed subsequent recruitment of RPA and RAD51 to repair DSBs and restart replication. More importantly, DNA2 expression was significantly increased in human cancers and its expression correlated with patient outcome. Our findings therefore indicate that enhanced activity of DSB resection likely constitutes one mechanism whereby precancerous and cancerous cells might alleviate replication stress. Cancer Res; 1–12. 2012 AACR. Introduction Tumorigenesis is a multistep process by which normal cells successively acquire genetic alterations (1). Analyses of human tumors have shown that the presence of DNA damage, particularly DNA double-strand breaks (DSB), distinguishes precancerous lesions and cancer fromnormal tissues (2, 3). Recent studies have indicated that oncogene-induced replication stress underlies DSB formation (4, 5). Specifically, activation of oncogenes provides cells with sustained proliferative signaling and leads to inappropriate DNA replication, which results in fork collapse and DSBs (6, 7). There are 2 pathways for repairing DSBs: homologous recombination (HR) and nonhomologous end-joining (NHEJ; ref. 8). NHEJ involves direct ligation of broken ends and primarily occurs in the G1 phase of the cell cycle. HR is considered to bemore error-free repairmechanism that copies sequences from the homologous template to repair damaged DNA. It predominantly occurs in the Sand G2 phases, when preferable template, sister chromatids, are available. Thereby, HR is a key pathway for repairing stalled and collapsed replication forks that occur spontaneously or are induced by topoisomerase I inhibitors such as camptothecin or polymerases inhibitors such as aphidicolin (9). Repair of chromosomal breaks by HR is initiated by resection of the 50 strands that generates 30 ssDNA tails at DSB ends (10, 11). Resection allows loading of single-strand–binding protein RPA that is further replaced by key enzyme in HR, strand exchange protein RAD51 with the help of BRCA2 (12–14). RAD51 mediates homology search and strand invasion at template sister chromatid, which is followed by DNA synthesis and resolution of recombination intermediate that restores replication fork. Depletion of RAD51 leads to accumulation of unrepaired DSBs and cells death showing the importance of HR for repair of spontaneous DNA breaks occurring during replication (15). Continuous formation of DSBs induced by replication stress activates DNA damage response (DDR), which induces senescence or apoptosis and thus prevents precancerous lesions from progressing to malignant lesions (6, 7). Impairment of DDR (e.g., through loss of expression of signaling kinases ATM or CHK2) can lead to a breach of this anti-cancer barrier and tumor progression (4, 5). However, it remains unclear that how precancerous and cancerous cells could cope with increased replication-associated DSBs and maintain their hyperactive DNA replication status. In this study, we used a proteomic approach to investigate protein components preferentially associated with replication forks in the presence of oncogene activation. Here, we report that human nuclease/helicase DNA2 is overexpressed in a variety of cancers and it plays an important role in alleviating Authors' Affiliations: Departments of Clinical Cancer Prevention, Systems Biology, and Surgical Oncology, Division of Surgery, The University of Texas MD Anderson Cancer Center; Institute of Biosciences and Technology, Texas A&M Health Science Center; and Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Authors: Guang Peng, Department of Clinical Cancer Prevention, Unit 1360, The University of Texas MD Anderson Cancer Center, Houston, TX 77054. Phone: 713-834-6151; Fax: 713-834-6397; E-mail: gpeng@mdanderson.org; and Shiaw-Yih Lin, Department of Systems Biology, Unit 950, The University of Texas MD Anderson Cancer Center, Houston, TX 77054. Phone: 713-563-4217; Fax: 713-563-4235; E-mail: sylin@mdanderson.org doi: 10.1158/0008-5472.CAN-11-3152 2012 American Association for Cancer Research. Cancer Research www.aacrjournals.org OF1 Research. on July 20, 2017. © 2012 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst April 9, 2012; DOI: 10.1158/0008-5472.CAN-11-3152