TY - JOUR
T1 - Partial reconstitution of human DNA mismatch repair in vitro
T2 - Characterization of the role of human replication protein A
AU - Ramilo, Cecilia
AU - Gu, Liya
AU - Guo, Shuangli
AU - Zhang, Xiping
AU - Patrick, Steve M.
AU - Turchi, John J.
AU - Li, Guo Min
PY - 2002
Y1 - 2002
N2 - 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.
AB - 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.
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U2 - 10.1128/MCB.22.7.2037-2046.2002
DO - 10.1128/MCB.22.7.2037-2046.2002
M3 - Article
C2 - 11884592
AN - SCOPUS:0036121372
SN - 0270-7306
VL - 22
SP - 2037
EP - 2046
JO - Molecular and cellular biology
JF - Molecular and cellular biology
IS - 7
ER -