DNA Mismatch Repair in Nuclear Extracts of Mouse M12 Cells

• Main Authors: Huang, Yao-ming, 黃耀民
• Other Authors: Woei-horng Fang
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/44291087368418285751

碩士 === 國立臺灣大學 === 醫事技術學研究所 === 88 === We constructed a set of DNA heteroduplexes contain eight possible base pair mismatches to characterize the repair activity in mouse cell extracts. We found that cell free nuclear extracts derived form mouse M12 cell line were able to correct single base-base mispairs within open circular DNA heteroduplexes containing a strand-specific, site-specific incision. Correction in this extract system, like human mismatch repair pathway, was strand specific, and highly biased to the incised DNA strand. The in vitro activity was dependent on ATP, dNTP, Mg2+, and the distance between the incision and mismatch site. Correction of each of these heteroduplexes was abolished by aphidicolin but was relatively insensitive to the presence of ddTTP, indicating probable involvement of DNA polymerase d in this system. Different base-base mismatches within a homologous set of heteroduplexes were processed with different efficiencies. Similar to bacterial and human system, the C-C heteroduplex was the weakest substrates. These results suggest that mouse cells possess a general, strand-specific mismatch repair system analogous to the Escherichia coli mutHLS and human mismatch repair system that contribute in a major way to the genetic stability. Finally, we established a reliable assay to examine the mismatch repair activity in mouse cells and this can be a tool to study the biochemical feature of mismatch repair in the future.