Thesis (M.Sc.). / Written for the Division of Experimental Medicine. Title from title page of PDF (viewed 2009/06/19). Includes bibliographical references.

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DNA is constantly damaged by a variety of exogenous and endogenous sources. To maintain the integrity of the genome, different DNA repair mechanisms have evolved, which deal with different kinds of DNA damage. One of the DNA repair pathways, Nucleotide Excision Repair (NER), is highly conserved throughout the three kingdoms of life and deals mainly with lesions arising in the DNA duplex after exposure to UV-light. The NER pathway in archaea is more closely related to that of eukarya, although the overall process is not yet well understood. This thesis describes the isolation and characterisation of one of the repair factors, XPD, from the crenarchaeon Sulfolobus acidocaldarius (SacXPD). SacXPD was first identified due to its homology with the eukaryal XPD protein. In eukarya XPD is the 5a' -> 3a' helicase involved in opening the DNA duplex around a damaged site. In eukarya, XPD is part of a 10-subunit complex, where it fulfils important structural roles and takes part in NER, transcription initiation from RNA polymerase II promoters and cell cycle regulation. The archaeal protein on the contrary is a monomer and a 5a' -> 3a' SF2 DNA helicase as its eukaryal counterpart. Its cellular functions, however, are unclear. Upon purification of SacXPD, it was discovered that the protein binds an ironsulphur cluster (FeS), which is essential for its helicase activity, but not for any other enzymatic functions, such as the ATP hydrolysing activity. The FeS cluster domain was not only identified in archaeal XPD, but also in eukaryal XPD and other related eukaryal helicases, such as FancJ. The presence of the FeS cluster was confirmed in the eukaryotic XPD homologue Rad3 from Saccharomyces cerevisiae. Mutagenesis studies were used to investigate a possible function of the FeS cluster, which may be used to engage ssDNA during the duplex unwinding process. This would actively distort the ss/ ds DNA junction. In addition, the resulting bending of the clamped single DNA strand could be used to avoid reannealing. The consequences of some human mutations introduced into the SacXPD gene were investigated and could contribute to our understanding of the development of human diseases.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 5, 2008). Includes bibliographical references.

Chromatin structure is intricately involved in the mechanisms of eukaryotic gene regulation. In general, the compact nature of chromatin blocks DNA accessibility such that components of the transcriptional machinery are unable to access regulatory sequences and gene activation is repressed. These repressive effects can be overcome or augmented by the actions of chromatin remodeling enzymes. Numerous studies highlight two classes of these enzymes: those that covalently modify nucleosomal histones and those that utilize energy derived from ATP hydrolysis to destabilize the histone-DNA contacts within the nucleosome (13, 14, 92). Members of each of these groups of chromatin remodeling enzymes play pivotal roles in modulating chromatin structure and in facilitating or blocking the binding of transcription factors. Mutations in genes encoding these enzymes can result in transcriptional deregulation and improper protein expression. Therefore, the regulation of chromatin structure is critical for precise regulation of almost all aspects of gene expression. Consequently, enzymes regulating chromatin structure are important modulators of cellular processes such as cell viability, growth, and differentiation. There remain many uncharacterized members of the ATP-dependent class of remodeling enzymes; characterization of these proteins will further elucidate the cellular functions these enzymes control. Here, we focus primarily on the ATP-dependent remodeling complexes, specifically the chromodomain helicase DNA-binding (CHD) family. The CHD proteins are distinguished from other ATP-dependent complexes by the presence of two N-terminal chromodomains that function as interaction surfaces for a variety of chromatin components. These proteins also contain a SNF2-like ATPase motif and are further classified based on the presence or absence of additional domains. Genetic, biochemical, and structural studies demonstrate that CHD proteins are important regulators of transcription and play critical roles during developmental processes. Numerous CHD proteins have also been implicated in human disease. The first CHD family member, mChd1, was identified in 1993 in a search for DNA-binding proteins with an affinity for immunoglobin promoters. Since then, additional CHD genes have been identified based on sequence and structural homology to mChd1. Despite an increase in the number of studies relating to CHD proteins, the function of most remains unknown or poorly characterized. Using embryonic stem (ES) cells containing an insertional mutation in the murine Chd2 locus, we generated a Chd2-mutant mouse model to address the biological effects of Chd2 in development and disease. The targeted Chd2 allele resulted in a stable Chd2-βgeo fusion protein that contained the tandem chromodomains, the SNF2-like ATPase motif, but lacked the C-terminal portion of the DNA-binding domain. We demonstrated that the mutation in Chd2 resulted in a general growth delay in homozygous mutants late in embryogenesis as well as perinatal lethality. Similarly, heterozygous mice showed a decreased neonatal viability. Moreover, the surviving heterozygous mice showed a general growth delay during the neonatal period and increased susceptibility to non-neoplastic lesions affecting multiple organs, most notably the kidneys. We further examined the connection between Chd2 and kidney disease in this murine model. Our findings revealed that the kidney phenotype observed in Chd2 mutant mice led to the development of membranous glomerulopathy, proteinuria, and ultimately to impaired kidney function. Additionally, serum analysis revealed decreased hematocrit levels in the Chd2-mutant mice, suggesting that the membranous glomerulopathy observed in these mice is associated with anemia. Lastly, we investigated whether the type of anemia observed in the Chd2-mutant mice. Red blood cell (RBC) indices and morphological examination of the RBCs indicated that the anemia seen in the Chd2-mutant mice can be classified as normocytic and normochromic. Further analyses have been initiated to determine if the anemia is due to an intrinsic effect in erythropoiesis or a secondary consequence of the glomerular disease. In summary, our findings have contributed to our understanding of the putative chromatin remodeling enzyme Chd2. Although much remains to be studied, these findings demonstrate a role for Chd2 in mammalian development and have revealed a link between Chd2 and disease.

The MCM helicase plays an important role in eukaryotic DNA replication, unwinding double stranded DNA ahead of the replication fork. MCM is a hetero-hexamer consisting of the six related proteins, Mcm2-Mcm7. The distantly related MCM-binding protein (MCM-BP) was first identified in a screen for proteins interacting with MCM2-7 in human cells and was found to specifically interact with Mcm3-7 but not Mcm2. It is conserved in most eukaryotes and seems to play an important role in DNA replication but its exact function is not clear yet. This study contributes to the understanding of the fission yeast homologue of MCM-BP, named Mcb1, but also of MCM-BP in general. Results presented in this thesis document the initial biochemical characterisation of the complex Mcb1 forms with Mcm proteins, the MCMᴹᶜᵇ¹ complex. Interactions of Mcb1 with Mcm proteins, potential interaction sites between the proteins and the size of the complex were analysed using a variety of methods, including tandem affinity purification, co-immunoprecipitation, sucrose gradients and in vitro pull-down assays. Sequence analysis and structure prediction were utilised to gain some insight into Mcb1 and MCM-BP ancestry and structure. Results presented here indicate that fission yeast Mcb1 shares homology with Mcm proteins and forms a complex with Mcm3-Mcm7 but not Mcm2 and thus replaces the latter in an alternative high molecular weight complex that is likely to have an MCM-like appearance. Deletion of mcb1⁺ showed that Mcb1 is essential in fission yeast. To examine the cellular function of the protein, temperature-sensitive mutants were generated. Inactivation of Mcb1 leads to an increase in DNA damage and cell cycle arrest in G2-phase depending on the activation of the Chk1 dependent DNA damage checkpoint. Similar observations were made when Mcb1 was overexpressed, indicating that certain levels of the protein are important for accurate DNA replication. Construction of truncated versions of Mcb1 suggested that almost the full-length protein is needed for proper function.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 82-92; 128-134). Online version available via ProQuest Digital Dissertations.

Tuberculosis, caused by the infection with Mycobacterium tuberculosis remained as a major global health challenge with one third of world population being infected by this pathogen. M. tuberculosis can persist for decades in infected individuals in the latent state as an asymptomatic disease and can emerge to cause active disease at a later stage. Thus, pathways and the mechanisms that are involved in the maintenance of genome integrity appear to be important for M. tuberculosis survival, persistence and pathogenesis. Helicases are ubiquitous enzymes known to play a key role in DNA replication, repair and recombination. However, role of helicases in providing selective advantage for M. tuberculosis survival and genome maintenance is obscure. Therefore, understanding the role of various helicases could provide insights into the M. tuberculosis survival, persistence and pathogenesis in humans. This information could be useful in considering helicases as a novel therapeutic target as well as developing effective vaccines. The research focus of my thesis has been to understand the role of helicases in safeguarding the M. tuberculosis genome from various genotoxic stresses. The major focus of the current study has been addressed towards understanding the role of M. tuberculosis RecG (MtRecG) helicase in recombinational repair and in remodeling stalled replication forks. This study highlights the importance of RecG helicase in the maintenance of genome integrity via DNA repair, recombination and in remodeling the stalled replication forks in M. tuberculosis. The thesis has been divided into following sections as follows: Chapter I: General introduction that describes the causes and consequences of replication stress and DNA repair pathways in M. tuberculosis The genome is susceptible to various types of damage induced by exogenous as well as endogenous DNA damaging agents. Unrepaired or misrepaired DNA lesions can lead to gross chromosomal rearrangements and ultimately cell death. Thus, organisms have evolved with efficient DNA damage response machinery to cope up with deleterious effects of genotoxic agents. Accurate transmission of genetic information requires error-free duplication of chromosomal DNA during every round of cell division. Defects associated with replication are considered as a major source of genome instability in all organisms. Normal DNA replication is hampered when the fork encounters road blocks that have the potential to stall or collapse a replication fork. The types of lesions that potentially block replication fork include lesions on the template DNA, various secondary structures, R-loops, or DNA bound proteins. To understand the DNA damage induced replication stress and the role of fork remodeling enzymes in the repair of stalled replication forks and its restart, chapter I of the thesis has been distributed into multiple sections as follows: Briefly, initial portion of the chapter describes overall replication process in prokaryotes highlighting the importance of coordinated replisome assembly and disassembly during initiation and termination. Later section discusses about various types of exogenous and endogenous DNA damages leading to replication fork stalling. Subsequent section of chapter I provide detailed description and mechanism of various repair pathways cell operates to repair such damages. Chapter I further summarizes causes of stalled replication forks majorly including template lesions, natural impediments like DNA secondary structures and DNA-protein cross links. Subsequent section discusses various pathways of replication restart that include essential role of primosomal proteins in reloading replisome machinery at stalled replication forks. Subsequent section of chapter I provide a comprehensive description of replication fork reversal (RFR) and mechanism of replication restart. RFR involves unwinding of blocked forks via simultaneous unwinding and annealing of parental and daughter strands to generate Holliday junction (HJ) intermediate. Genetic and biochemical studies highlighted the importance of RecG, RuvAB and RecA proteins in driving RFR reaction in E. coli. Hence, in the subsequent chapter, the functional role of RecG, RuvAB and RecA in replication-recombination processes has been discussed. Last section of the chapter devotes completely to M. tuberculosis, its genome dynamics and the various pathways of mycobacterial DNA repair. M. tuberculosis experiences substantial DNA damage inside host macrophages owing to the acidic environment, reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) which are sufficient enough to cause replication stress. To gain insights into the role of M. tuberculosis RecG helicase in DNA repair, recombination and in remodeling the stalled replication forks the following objectives were laid for my PhD thesis: 1 To understand the functional role of M. tuberculosis RecG (MtRecG) in DNA repair and recombination. 2 To investigate the distinct role(s) of MtRecG, MtRuvAB and MtRecA in remodeling the stalled replication forks. Chapter II: Evidence for the role of Mycobacterium tuberculosis RecG helicase in DNA repair and recombination In order to survive and replicate in a variety of stressful conditions during its life cycle, M. tuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. Helicases are one such ubiquitous enzyme involved in all DNA metabolic transaction pathways for maintenance of genome stability. To understand the role of M. tuberculosis RecG (MtRecG) helicase in recombination and repair, we carried out functional and biochemical studies. In our study, we show that M. tuberculosis RecG expression was induced in response to different genotoxic agents. Strikingly, expression of M. tuberculosis RecG in Escherichia coli ∆recG mutant strain provided protection against MMC, MMS and UV-induced cell death. Purified M. tuberculosis RecG exhibited higher binding affinity for the Holliday junction (HJ) as compared to a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile junction, branch migration and resolution was evident only in the case of the mobile junction. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently as compared to the mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structures, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Altogether, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as in stress conditions. Chapter III: Mycobacterium tuberculosis RecG but not RuvAB or RecA is efficient at remodeling the stalled replication forks: Implications for multiple mechanisms of replication restart in mycobacteria Aberrant DNA replication, defects in the protection and restart of stalled replication forks are a major cause of genome instability in all organisms. Replication fork reversal is emerging as an evolutionarily conserved physiological response for restart of stalled forks. Escherichia coli RecG, RuvAB and RecA proteins have been shown to reverse the model replication fork structures in vitro. However, the pathways and the mechanisms by which Mycobacterium tuberculosis, a slow growing human pathogen responds to different types of replication stress and DNA damage is unclear. In our study, we show that M. tuberculosis RecG rescues E. coli ∆recG cells from replicative stress. The purified M. tuberculosis RecG (MtRecG) and RuvAB (MtRuvAB) proteins catalyze fork reversal of model replication fork structures with and without leading strand ssDNA gap. Interestingly, SSB suppresses the MtRecG and MtRuvAB mediated fork reversal with substrates that contain lagging strand gap. Notably, our comparative studies with fork structures containing template damage and template switching mechanism of lesion bypass reveal that MtRecG but not MtRuvAB or MtRecA is proficient in driving the fork reversal. Finally, unlike MtRuvAB, we find that MtRecG drives efficient reversal of forks when fork structures are tightly bound by protein. These results provide direct evidence and valuable insights into the underlying mechanism of MtRecG catalyzed replication fork remodeling and restart pathways in vivo.

Faithful duplication and segregation of undamaged DNA is critical to the survival of all organisms and prevention of oncogenesis in multicellular organisms. To ensure inheritance of intact DNA, cells rely on checkpoints. Checkpoints alter cellular processes in the presence of DNA damage preventing cell cycle transitions until replication is completed or DNA damage is repaired. Several checkpoints are specific to S-phase. The S-M replication checkpoint prevents mitosis in the presence of unreplicated DNA. Rather than outright halting replication, the S-phase DNA damage checkpoint slows replication in response to DNA damage. This checkpoint utilizes two general mechanisms to slow replication. First, this checkpoint prevents origin firing thus limiting the number of replication forks traversing the genome in the presence of damaged DNA. Second, this checkpoint slows the progression of the replication forks. Inhibition of origin firing in response to DNA damage is well established, however when this thesis work began, slowing of replication fork progression was controversial. Fission yeast slow replication in response to DNA damage utilizing an evolutionarily conserved kinase cascade. Slowing requires the checkpoint kinases Rad3 (hATR) and Cds1 (hChk2) as well as additional checkpoint components, the Rad9-Rad1-Hus1 complex and the Mre11-Rad50-Nbs1 (MRN) recombinational repair complex. The exact role MRN serves to slow replication is obscure due to its many roles in DNA metabolism and checkpoint response to damage. However, fission yeast MRN mutants display defects in recombination in yeast and, upon beginning this project, were described in vertebrates to display S-phase DNA damage checkpoint defects independent of origin firing. Due to these observations, I initially hypothesized that recombination was required for replication slowing. However, two observations forced a paradigm shift in how I thought replication slowing to occur and how replication fork metabolism was altered in response to DNA damage. We found rhp51Δ mutants (mutant for the central mitotic recombinase similar to Rad51 and RecA) to slow well. We observed that the RecQ helicase Rqh1, implicated in negatively regulating recombination, was required for slowing. Therefore, deregulated recombination appeared to actually be responsible for slowing failures exhibited by the rqh1Δ recombination regulator mutant. Thereafter, I began a search for additional regulators required for slowing and developed the epistasis grouping described in Chapters II and V. We found a wide variety of mutants which either completely or partially failed to slow replication in response to DNA damage. The three members of the MRN complex, nbs1Δ, rad32Δ and rad50Δ displayed a partial defect in slowing, as did the helicase rqh1Δ and Rhp51-mediator sfr1Δ mutants. We found the mus81Δ and eme1Δ endonuclease complex and the smc6-xhypomorph to completely fail to slow. We were able to identify at least three epistasis groups due to genetic interaction between these mutants and recombinase mutants. Interestingly, not all mutants’ phenotypes were suppressed by abrogation of recombination. As introduced in Chapters II, III and IV checkpoint kinase cds1Δ, mus81Δ endonuclease, and smc6-x mutant slowing defects were not suppressed by abrogation of recombination, while the sfr1Δ, rqh1Δ, rad2Δ and nbs1Δ mutant slowing defects were. Additionally, data shows replication slowing in fission yeast is primarily due to proteins acting locally at sites of DNA damage. We show that replication slowing is lesion density-dependent, prevention of origin firing representing a global response to insult contributes little to slowing, and constitutive checkpoint activation is not sufficient to induce DNA damage-independent slowing. Collectively, our data strongly suggest that slowing of replication in response to DNA damage in fission yeast is due to the slowing of replication forks traversing damaged template. We show slowing must be primarily a local response to checkpoint activation and all mutants found to fail to slow are implicated in replication fork metabolism, and recombination is responsible for some mutant slowing defects.

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