Studies of DNA repair and mutagenesis in Escherichia coli and Bacillus subtilis
Author(s)Smith, Bradley Theodore
Massachusetts Institute of Technology. Dept. of Biology.
Graham C. Walker.
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All organisms face constant challenges to the integrity of their genomes, from environmental agents such as ultraviolet light (UV) to errors generated by their own DNA polymerases. To deal with these challenges, all organisms possess a range of DNA repair and damage tolerance systems. In bacteria, there exists an inducible response to DNA damage termed the SOS response, in which damage induces the expression of greater than forty genes involved in repair. Mismatch repair (MMR) is a system by which the cell is able to avoid mutations by correcting DNA replication errors. Working with living Bacillus subtilis cells, the studies described here have found that MutS and MutL, the key proteins of MMR, form discrete foci in response to mismatches. These MMR foci are the active sites of repair, and appear to assemble near the DNA polymerase at mismatched base-pairs. This is consistent with a model in which the newly-synthesized DNA strand containing the error is discriminated from the parental strand via interactions between MMR proteins and the replisome.(cont.) Nucleotide excision repair (NER) is a damage inducible system that can act throughout the genome to repair a wide range of damage. These studies have found that UvrA, the damage recognition protein of NER, is associated with the entire B. subtilis chromosome both before and after damage, suggesting that the UvrA²B damage recognition complex is constantly scanning the genome. In addition, the chromosome was observed to undergo a damage-induced reconfiguration that was dependent on the SOS response and was also reversible after low doses of damage. Finally, these studies have found that the products of the SOS-regulated umuDC operon in Escherichia coli may be involved in a primitive DNA damage checkpoint that acts after damage to slow the resumption of DNA replication, allowing additional time for repair to occur. It was found that the protection provided by this putative checkpoint was dependent on an active NER system. This checkpoint activity was found to be independent of the traditional role of the umuDC operon in the process of SOS mutagenesis.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2001.Includes bibliographical references.
DepartmentMassachusetts Institute of Technology. Dept. of Biology.
Massachusetts Institute of Technology