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Mechanism of Replicative DNA Polymerase Delta Pausing and a Potential Role for DNA Polymerase Kappa in Common Fragile Site Replication
Authors:Erin Walsh  Xiaoxiao Wang  Marietta Y Lee  Kristin A Eckert
Institution:1. Cellular and Molecular Biology Graduate Program, Penn State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA;2. Department of Pathology, Jake Gittlen Cancer Research Foundation, Penn State University College of Medicine, 500 University Drive, Mailcode H059, Hershey, PA 17033, USA;3. Department of Biochemistry and Molecular Biology, New York Medical College, 40 Sunshine Cottage Road, Valhalla, NY 10595, USA;1. Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, China;2. National Engineering and Research Center of Human Stem Cell, Changsha, China;3. Key Laboratory of Stem Cells and Reproductive Engineering, Ministry of Health, Changsha, China;4. Reproductive & Genetic Hospital of CITIC-Xiangya, Changsha, China;1. Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Washington University School of Medicine, Siteman Cancer Center, St. Louis, MO, United States;2. NRG Oncology Statistics and Data Management Center, Roswell Park Cancer Institute, Buffalo, NY, United States;3. Division of Gynecologic Oncology (FB), Anatomic Pathology (NCR), Obstetrics and Gynecology (PG), Ohio State University, Columbus, OH, United States;4. Gynecologic Oncology, Stephenson Oklahoma Cancer Center, Oklahoma City, OK, United States;5. Gynecologic Oncology, Women and Infants Hospital, Providence, RI, United States;6. Gynecologic Oncology, University of Colorado Cancer Center, Aurora, CO, United States;7. Gynecologic Oncology, University Hospital Case Medical Center, Cleveland, OH, United States;8. Gynecologic Oncology, University of Minnesota Medical School, Minneapolis, MN, United States;9. Gynecologic Oncology, Stony Brook University Hospital, Stony Brook, NY, United States
Abstract:Common fragile sites (CFSs) are hot spots of chromosomal breakage, and CFS breakage models involve perturbations of DNA replication. Here, we analyzed the contribution of specific repetitive DNA sequence elements within CFSs to the inhibition of DNA synthesis by replicative and specialized DNA polymerases (Pols). The efficiency of in vitro DNA synthesis was quantitated using templates corresponding to regions within FRA16D and FRA3B harboring AT-rich microsatellite and quasi-palindrome (QP) sequences. QPs were predicted to form stems of ~ 75–100% self-homology, separated by 3–9 bases of intervening sequences. Analysis of DNA synthesis progression by human Pol δ demonstrated significant synthesis perturbation both at A]n and TA]n repeats in a length-dependent manner and at short (< 40 base pairs) QP sequences. DNA synthesis by the Y-family polymerase κ was significantly more efficient than Pol δ through both types of repetitive elements. Using DNA trap experiments, we show that Pol δ pauses within CFS sequences are sites of enzyme dissociation, and dissociation was observed in the presence of RFC-loaded PCNA. We propose that enrichment of microsatellite and QP elements at CFS regions contributes to fragility by perturbing replication through multiple mechanisms, including replicative Pol pausing and dissociation. Our finding that Pol δ dissociates at specific CFS sequences is significant, since dissociation of the replication machinery and inability to efficiently recover the replication fork can lead to fork collapse and/or formation of double-strand breaks in vivo. Our biochemical studies also extend the potential involvement of Y-family polymerases in CFS maintenance to include polymerase κ.
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