Partial Reconstitution of Human DNA Mismatch Repair In Vitro: Characterization of the Role of Human Replication Protein A

• DOI: 10.1128/mcb.22.7.2037-2046.2002
• Publication Date: 2002-07-27T09:59:40Z
• Authors: Cecilia Ramilo; Liya Gu; Shuangli Guo; Xiping Zhang; Steve M. Patrick; John J. Turchi; Guo-Min Li
• Keywords: Replication; Replication protein A

AbstractDNA mismatch repair (MMR) is a critical genome-stabilization system. However, the molecular mechanism of MMR in human cells remains obscure because many components have not yet been identified. Using functional vitro reconstitution system, this study identified three HeLa cell fractions essential for MMR. These divide into two distinct stages: mismatch-provoked excision and synthesis. In dissection reaction crucial intermediates elucidated biochemical functions individual hitherto unknown replication protein A (hRPA) Thus, one fraction carries out nick-directed mismatch-dependent excision; second DNA synthesis ligation; third provides hRPA, which plays multiple roles by protecting template strand from degradation, enhancing excision, facilitating It anticipated that further analysis these will identify additional enable complete pathway with purified proteins. We thank Peggy Hsieh (National Institute Diabetes Digestive Kidney Diseases) comments on manuscript, Zhengxiang Pan (Mount Sinai School Medicine) hRPA antibodies, Jianxin Wu Scott McCulloch help phosphocellulose chromatography, Lu Qiu nuclear extract preparations.This work was supported part grants CA82604 CA85377 (to G.-M.L.) CA82741 J.J.T.) National Cancer Institute.