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

Abstract

DNA mismatch repair (MMR) is a critical genome-stabilization system. However, the molecular mechanism of MMR in human cells remains obscure because many of the components have not yet been identified. Using a functional in vitro reconstitution system, this study identified three HeLa cell fractions essential for in vitro MMR. These fractions divide human MMR into two distinct stages: mismatch-provoked excision and repair synthesis. In vitro dissection of the MMR reaction and crucial intermediates elucidated biochemical functions of individual fractions in human MMR and identified hitherto unknown functions of human replication protein A (hRPA) in MMR. Thus, one fraction carries out nick-directed and mismatch-dependent excision; the second carries out DNA repair synthesis and DNA ligation; and the third provides hRPA, which plays multiple roles in human MMR by protecting the template DNA strand from degradation, enhancing repair excision, and facilitating repair synthesis. It is anticipated that further analysis of these fractions will identify additional MMR components and enable the complete reconstitution of the human MMR pathway with purified proteins.

We thank Peggy Hsieh (National Institute of Diabetes and Digestive and Kidney Diseases) for comments on the manuscript, Zhengxiang Pan (Mount Sinai School of Medicine) for hRPA antibodies, Jianxin Wu and Scott McCulloch for help in phosphocellulose chromatography, and Lu Qiu for nuclear extract preparations.

This work was supported in part by grants CA82604 and CA85377 (to G.-M.L.) and CA82741 (to J.J.T.) from the National Cancer Institute.