During the course of normal DNA replication, replication forks are constantly encountering "housekeeping" types of routine damage to the DNA template that may cause the forks to stall or collapse. One product of this fork collapse is the induction of the SOS response, a coordinated global response to help pause the growth and replication of a cell while DNA damage is addressed and repaired. In E. coli, this response is activated by the formation of ssDNA, to which the RecA protein binds and forms a nucleoprotein filament, which acts as the activator for autocleavage of the LexA transcriptional repressor, which normally represses expression of SOS genes. Damage responses are crucial to maintaining genomic integrity, and are therefore essential to all forms of life, and this type of regulatory system is highly conserved. However, cells have mechanisms for tightly regulating induction of these responses, and can often repair routine damage to their chromosomes without the need to induce SOS. This is chiefly evidenced by the observation that more than 20% of cells in a population have RecA filaments, but less than 1% are induced for SOS. How cells make this decision to induce SOS is the subject of this work. This dissertation describes three projects aimed at examining molecular mechanisms by which cells regulate RecA filaments, and therefore the decision to induce the SOS response. The first examines the disparity between the formation of RecA filaments, as evidenced by RecA-GFP foci, and the induction of SOS in the absence of damage, using a psulA-gfp reporter system. It is shown that there are three independent factors that repress SOS expression in undamaged E. coli cells. These are radA, the amount of recA in the cell, and in some circumstances recX. The first two limit SOS in wild type cells in the absence of external damage, while the third is an additional factor required in xthA mutants, likely due to the fact there are more RecA loading events in these mutants. These factors are thought to change the character and reduce the half-life and persistence of RecA filaments in the cell. The second project shows that suppression of SOS through the use of recA4162 and uvrD303 mutants is substrate and situation-specific. This specificity is demonstrated by the fact that, while both recA4162 and uvrD303 can suppress SOS in the SOS constitutive mutant recA730, recA4162 can only suppress SOS when the signal occurs at replication forks and not at any other place on the chromosome, while uvrD303 appears to suppress SOS with less specificity, and can suppress after UV (shown previously), at induced DSBs, and other places not directly at the replication fork. Here mutants of different replication factors are used that uncouple the replisome and induce SOS to a high degree. The third project determines the factors necessary for loading RecA filaments at the replication fork versus other locations on the chromosome when SOS is induced in the absence of damage, and helps elucidate further mechanisms for induction of SOS at these substrates. It is shown that the sbcB and recJ exonucleases assist in inappropriate RecA filament formation by substrate processing exclusively at replication forks, but not other substrates, likely through mechanisms that are reliant on the activities of the RecA loading factors RecBCD and RecFOR.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-6882 |
| Date | 01 January 2013 |
| Creators | Massoni, Shawn Christopher |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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