Mechanistic Study of Hairpin DNA Processing in Escherichia coli Extracts

• Main Authors: Yu-Ting Chang, 張友婷
• Other Authors: Woei-horng Fang
• Format: Others
• Language: en_US
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/99022257921640592894

碩士 === 國立臺灣大學 === 醫事技術學研究所 === 91 === Tandem repeat or palindromic sequences present in DNA may form secondary structures that block DNA replication and transcription causing adverse effects on genome stability. Secondary structures such as hairpin-loops have been shown to be associated with a number of human diseases, such as myotonic dystrophy, Huntington’s disease, and fragile X syndrome. Escherichia coli possesses a methyl-directed mismatch repair (MMR) pathway responsible for correcting biosynthetic errors and ensuring the fidelity of homologous genetic recombinations. But MMR pathway can only repair base-base mismatches and loops of small heterologies. Larger loops are rectified predominately by a loop repair pathway that could repair heterologies of several hundreds of base pairs. It has been suggested that hairpin structures containing mispaired bases could stimulate the E. coli MMR system. To investigate this possibility, we constructed a set of DNA heteroduplexes containing mispairs within hairpin structures to characterize the in vitro reaction in Escherichia coli cell extracts. Our results show that hairpin structures are repaired through a pathway independent of MMR since repair reactions occurred in the absence of the mismatch repair gene products MutS, MutL, and MutH. Mismatch-containing hairpins have higher repair efficiencies and the shorter hairpin (10-bp) is repaired most efficiently. The SbcCD protein which is known to cleave hairpin structures that occurred during DNA replication does not take part in hairpin repair in our assay system. Similar to the loop repair pathway, a strand break located either 3’ or 5’ to the loop is sufficient to activate hairpin repair to the nicked strand in Escherichia coli extracts. This system only requires Mg2+ and the four dNTPs for efficient repair on both insertions and deletions.