Homologous recombination is generally considered a major mechanism by which cells repair many types of DNA lesions and damaged replication forks. However, if this process is left unchecked, cells often show a hyper-recombination (hyper-rec) phenotype, and are susceptible to large deletions, duplications, or inversions of important genetic information. This dissertation describes two projects aimed at examining molecular mechanisms by which cells regulate homologous recombination. The first shows several 3'-5' exonucleases prevent RecA-GFP loading by destroying potential substrates. It is shown that two genetic pathways exist: one consisting of ExoIII and another comprised of ExoVII, ExoIX, ExoX, and ExoXI. ExoI acts upstream of both of these pathways. Although xthA cells have an increase in DSBs and recB-dependent loading of RecA-GFP, they are viable with a recB mutation and do not display a large increase in SOS expression. The increase in RecA-GFP is also independent of base excision repair (BER). These experiments uncovered that DNA in a population of wild type cells undergoes DSBs and is often repaired in a RecA-independent manner after processing by ExoI and ExoIII. The second project shows the helicase, UvrD limits the number and intensities of RecA-GFP foci. This activity is due to the ability of UvrD to remove RecA from DNA where it is loaded in a RecF pathway-dependent manner. This activity requires ATP binding by UvrD, suggesting that helicase/translocase activity is important for RecA-removal. The hyper-helicase mutation, uvrD303 confers UV sensitivity to cells. Epistasis analyses showed uvrD303 is defective in the recA pathway of UV repair and not in nucleotide excision repair (NER). Surprisingly, UvrD303 does not directly remove RecA after UV, as new RecA-GFP foci appear like in wild type cells. UvrD303 does, however, slightly inhibit SOS induction, and constitutively activating the SOS response restores UV resistance to these cells in a way that is independent of recA overexpression. Furthermore, uvrD303 was capable of suppressing the constitutive SOS phenotype of recA730. These experiments suggested that UvrD303 antagonizes the ability of RecA filaments to induce the SOS response, rendering cells UV sensitive.

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.

During the course of DNA replication, replication forks often stall or collapse as they proceed from oriC to the terminus due to housekeeping types of DNA damage or proteins bound to DNA. If the DNA is not repaired or if the replication forks do not restart, viability of the cell then becomes compromised. In Escherichia coli, if DNA damage is detected, approximately 40 genes are expressed to repair the offending DNA lesion. This is known the SOS response. Two proteins RecA and LexA regulate the SOS response, where RecA (when bound to ssDNA), serves as the sensor for DNA damage and LexA serves the repressor for SOS expression. When the RecA nucleoprotein filament forms, this complex will accelerate autocleavage of LexA inducing the response. Recently it has been observed that in a population of cells approximately 15% of the population had RecA bound to DNA, however at any given time approximately 0.3% of the population is induced for SOS expression suggesting that the cell can decide whether induce the SOS response or not. The aim of this work is to understand how the cell decides whether or not to express the SOS response at housekeeping types of DNA damage. Regulation is important because the cell would not want to express the SOS response every time replication forks encounter housekeeping types of DNA damage. The first component of this work looks at SOS expression in populations of cells during log phase growth using the fluorecense microscopy and the transcriptional fusion sulA-gfp. Results show that a SOS expression is stochastic and occurs in a small population of wild type cells. The second component of my work focuses on how the cell decides when to express the SOS response by using recA constitutive mutants that are defective in this regulation. Results show that the concentration and conformation of the RecA nucleoprotein filament is crucial for this to occur. Lastly novel recA mutants were created and examined for their role in suppressing constitutive SOS expression. It is observed that suppression of constitutive SOS expression could be seen when these mutations were supplied in cis and in trans, suggesting multiple levels of SOS regulation.

Transcription regulation in eukaryotes is a complex process governed by the concerted action of different factors. The work in this thesis is focused on transcriptional regulation in Saccharomyces cerevisiae. I analyzed the regulation of the phospholipid biosynthetic gene INO1 , which has been a model gene for transcription studies for over three decades. Some major questions that I have addressed are: what kinds of cis regulatory sequences and trans factors are important for regulation of INO1? What is the sequence of events in this regulation? How is the recruitment of these trans factors consequential for INO1 transcription? I present my results here for the role of the basic helix loop helix transcription factor (bHLH) family in coordinated regulation of INO1 transcription. I report that the centromeric binding factor 1 (Cbf1p) together with two other members of the bHLH protein family, Ino2p and Ino4p, are required for efficient derepression of INO1 transcription. Together these bHLH transcription factors recruit the ISW2 chromatin-remodeling complex onto the INO1 promoter to drive productive transcription from the INO1 locus. My efforts in studying the regulation of INO1 led me to study the regulation of SNA3, a gene found in tandem upstream (→→) to the INO1 gene and regulated by the same environmental conditions as INO1. Studies on the mechanism of coregulation of adjacent genes in budding yeast have been largely speculative. I provide evidence that the same bHLH proteins which regulate INO1 also regulate SNA3, albeit differentially. Significantly, my results also show that the regulation of both SNA3 and INO1 is dictated from the intergenic region between the two genes. This is a novel mechanism of transcription regulation in yeast as regulation from downstream of ORF is unknown in yeast. Thus, my results with both SNA3 and INO1 provide novel details on how the process of transcription is regulated in response to an environmental cue.

Polyhydroxyalkanoates (PHAs) comprise a family of macromolecules produced by many bacteria as a carbon and energy reserve and are perceived to have commercial potential as biodegradable thermoplastics. To investigate the biogenesis of PHA inclusion-bodies and the functions of inclusion-body proteins from Bacillus megaterium strain 11561, we identified and cloned a 7.9 kb DNA fragment harboring five genes, phaP, -Q, -R, -B, and -C specifying proteins having known or putative functions in PHA metabolism and/or inclusion-body biogenesis. Sequence similarities to known pha genes identified phaB and -C as specifying acetoacetyl-CoA reductase and PHA synthase, respectively. Putative proteins encoded by phaP, -Q, and -R were not ascribed functions due to lack of significant similarities to known proteins. Both the functionality of the pha gene cluster with respect to PHA accumulation and the transcriptional organization of the genes were determined. Subsequent studies were carried out to further investigate functions of phaP, -Q, and -R. PhaP was established as a major PHA inclusion-body associated protein and was shown to localize to inclusion-bodies in living cells. Further, we demonstrated a phasin-like role for this protein due to its affect on the formation of PHA inclusion-bodies. In addition, our data is consistent with PhaP functioning as a storage protein, implying that the role of PHA inclusion-bodies may be that of a reserve of amino acids in addition to reduced carbon. Regulation of phaP was influenced by PhaQ We showed that PhaQ is a transcriptional repressor of phaP. Moreover, we demonstrated the binding of PhaQ to inclusion-bodies, suggesting that its mode of regulation may involve its localization. Similarly, we showed that PhaR is bound to PHA inclusion-bodies. Our data demonstrated the requirement of phaR for PHA accumulation in vivo and that both PhaC and PhaR were necessary for PHA synthase activity in vitro. Evidence suggests that PHA synthase from strain 11561 can exist in an active or inactive state and that this state is either directly or indirectly influenced by PhaR. A working model is proposed to describe the roles of PhaP, -Q and -R in the metabolism of PHA and biogenesis of PHA inclusion-bodies.