Partial reconstitution of human DNA mismatch repair in vitro: Characterization of the role of human replication protein A
Partial reconstitution of human DNA mismatch repair in vitro: Characterization of the role of human replication protein A
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DOI：
10.1128/mcb.22.7.2037-2046.2002
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发表时间：
2002-04-01
影响因子：
5.3
通讯作者：
Li, GM
中科院分区：
文献类型：
--
作者：
Ramilo, C;Gu, LY;Li, GM
DNA错配修复(MMR)是一个重要的基因组稳定系统。然而，MMR在人类细胞中的分子机制仍然不清楚，因为许多成分还没有确定。利用体外功能重建系统，本研究鉴定了体外MMR所必需的三个HeLa细胞组分。这些片段将人类MMR分为两个不同的阶段：错配引起的切除和修复合成。对MMR反应和关键中间体的体外解剖阐明了人MMR中各个组分的生化功能，并确定了迄今未知的人类复制蛋白A(HrpA)在MMR中的功能。因此，一个片段进行缺口定向和错配依赖的切除；第二个片段进行DNA修复合成和DNA连接；第三个片段提供HrpA，它在人类MMR中发挥多种作用，保护模板DNA链不被降解，促进修复切除，并促进修复合成。预计对这些组分的进一步分析将确定更多的MMR组分，并使人类MMR途径能够与纯化的蛋白质完全重建。
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.