Repair Patch Analysis of Large Loop Repair
Date Issued
2008
Date
2008
Author(s)
Wei, Ting-Yu
Abstract
DNA loop heterologies are products of DNA polymerase slippage during DNA replication. Such DNA loop heterologies may lead to genomic instability if unrepaired. Previous results have shown that loop repair is nick-directed and independent of the mismatch repair proteins. However, the mechanism of large loop repair is still not clear. To investigate the repair process of large DNA loop, I analyed the repair patch of large loop repair. In the first part, I treated loop heterologies with HeLaS3 human nuclear cell extract to repair large DNA loop in vitro. I trapped repair intermediates by omitting dNTPs. I also did the repair patch mapping by using thiophosphate chemistry by addition of dNTPαS in the reaction. The repair products and intermediates were resolved by alkaline agarose gel electrophoresis and indirect labeling by southern blotting. These results showed that large loop repair is blocked when dNTPs is limited, and the repair reaction is limited to nick when dNTPαS is added. In the second part, I used E. coli cell extracts to study bacterial loop repair process. I replaced dTTP to dUTP in repair reaction, and treated DNA loop substrate with extract of BD2314, a ung- stain of E. coli, then used uracil glyscosylase and AP endonuclease to remove U and broke the phosphate bond. The breakage point was resolved on alkaline agarose gel. These data showed that the repair patch is between loop and nick. I further fine mapped the breakage sites by denaturing polyacylamide gel electrophoresis. I found that the repair synthesis is localized within the loop region. These data indicated that large loop repair in E. coli is nick-directed, and the base excison and resynthesis is localized around the loop.
Subjects
large loop repair
repair path
dUTP
alkaline agarose gel
polyacrylamide gel
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