Partial reconstitution of human DNA mismatch repair in vitro: characterization of the role of human replication protein A.
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.
Ramilo
Cecilia
C
Department of Pathology and Laboratory Medicine, Lucille P. Markey Cancer Center, University of Kentucky Medical Center, Lexington, Kentucky 40536, USA.
Gu
Liya
L
Guo
Shuangli
S
Zhang
Xiping
X
Patrick
Steve M
SM
Turchi
John J
JJ
Li
Guo-Min
GM
D020137:Base Pair Mismatch / Q000235:genetics*; D015153:Blotting, Western; D002457:Cell Extracts; D002467:Cell Nucleus / Q000201:enzymology; D002474:Cell-Free System; D004247:DNA / Q000378:metabolism; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051716:Replication Protein A; D013698:Templates, Genetic
D002457:Cell Extracts; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D011088:DNA Ligases