Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 67-74). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D.) - University of St Andrews, January 2007.

Thesis (Ph. D.)--Cornell University, 1989. / Vita. Includes bibliographical references.

Natural killer cells are innate immune effectors that kill virally infected or malignant cells. Natural killer cell deficiency (NKD) occurs when NK cell development or function are impaired, and individuals with NKD are susceptible to severe and recurrent viral infections. Several gene deficiencies result in NKD, including variants in MCM4, GINS1, MCM10 and GINS4, which are components of the CDC45-MCM-GINS (CMG) helicase. The CMG helicase unwinds DNA during replication and is expressed in any actively proliferating cell. NK cells are more strongly impacted by mutational deficiencies in helicase proteins than other lymphocytes, though the mechanisms underlying this susceptibility are not completely understood. NK cells from individuals with NKD as a result of helicase deficiency have increased DNA damage, cell cycle arrest, and replication stress. We found that activated NK cells undergo apoptosis and autophagy in response to this stress, unlike activated T cells. We also identified a patient with a damaging variant in CDC45 to further support these findings of the effects of replication stress on NK cells. This individual, due to broader involvement of the immune system, requires a wider definition of natural killer cell disease, termed NK IEI. However, this CDC45-deficient individual’s cells display disrupted cell cycle, increased DNA damage and replication stress, with upregulation of apoptosis genes in NK cells. These findings show that sensitivity to replication stress affects human NK cell survival and function and can contribute to NK cell deficiency and human disease.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

DNA is a reactive and dynamic molecule that is continually damaged by both exogenous and endogenous agents. Various DNA repair pathways have evolved to ensure the faithful replication of the genome. One such pathway, nucleotide excision repair (NER), involves the concerted action of several proteins to repair helix-distorting lesions that arise following exposure to UV light. Mutation of NER proteins is associated with several genetic diseases, including xeroderma pigmentosum that can arise upon mutation of the DNA helicase, XPD. The consequences of introducing human mutations into the gene encoding XPD from Sulfolobus acidocaldarius (SacXPD) were investigated to shed light on the molecular basis of XPD-related diseases. XPD is a 5’-3’ DNA helicase that requires an iron-sulphur (FeS) cluster for activity (Rudolf et al., 2006). Several proteins related to SacXPD, including human XPD, human FancJ and E. coli DinG, also rely on an FeS cluster for DNA unwinding (Rudolf et al., 2006; Pugh et al., 2008; Ren et al., 2009). Sequence analysis of the homologous protein, DinG, from Staphylococcus aureus (SarDinG) suggests that this protein does not encode a FeS cluster. In addition, SarDinG comprises an N-terminal extension with homology to the epsilon domain of polymerase III from E. coli. This thesis describes the purification and characterisation of SarDinG. During replication, DNA lesions or other ‘roadblocks’, such as DNA-bound proteins, can lead to replication fork stalling or collapse. To maintain genomic integrity, the fork must be restored and replication restarted. In archaea, the DNA helicase Hel308 is thought to play a role in this process by removing the lagging strands of stalled forks, thereby promoting fork repair by homologous recombination. Potential roles of Hel308 during replication fork repair are discussed in this thesis. The mechanism by which Hel308 moves along and unwinds DNA was also investigated using a combined structural and biophysical approach. The exchange of DNA between homologous strands, catalysed by a RecA family protein (RecA in bacteria, RAD51 in eukaryotes, and RadA in archaea), defines homologous recombination. While bacteria encode a single RecA protein, both eukaryotes and archaea encode multiple paralogues that have implications in the regulation of RAD51 and RadA activity, respectively. This thesis describes the purification and characterisation of one of the RadA paralogues (Sso2452) in archaea.

<p>The genetic information necessary for the survival and propagation of a species is contained within a physical structure, DNA. However, this molecule is sensitive to damage arising from both exogenous and endogenous sources. DNA damage can prevent metabolic processes such as replication and transcription; thus, systems to bypass or repair DNA lesions are essential. One type of lesion in particular - the double strand break (DSB) - is extremely dangerous as inappropriate repair of DSBs can lead to deletions, mutations and rearrangements. Homologous recombination (HR) uses a template with sequence homology to the region near the DSB to restore the damaged molecule. However, this high-fidelity pathway can contribute to genome instability when recombination occurs between diverged substrates. To further our understanding of the regulation of HR during vegetative growth, we have used the budding yeast Saccharomyces cerevisiae as a model system and a plasmid-based assay to model repair of a DSB. In the first part of this work, the molecular structures of noncrossover (NCO) and crossover (CO) products of recombination were examined. While the majority of NCOs had regions of heteroduplex DNA (hDNA) on one side of the gap in the repaired allele and no change to the donor allele, most COs had two tracts of hDNA. They were present on opposite sides of the gap, one in each allele. Our results suggest that the majority of NCOs are generated through synthesis-dependent strand annealing (SDSA), and COs are the result of constrained cleavage of a Holliday junction (HJ) intermediate. To clarify the mechanisms regulating NCO production, the effects of three DNA helicases - Mph1, Sgs1 and Srs2 - on the structures of NCO events were examined. All three helicases promote NCO formation by SDSA, but Sgs1 and Srs2 also assist in NCO formation arising from an HJ-containing intermediate, consistent with HJ-dissolution. To study how CO products are generated, we have investigated the contribution of the following candidate HJ resolvases to the structures of CO events: Mus81, Yen1 and Rad1. The results suggest that Rad1 is important to normal CO formation in this assay, but Mus81 and Yen1 are largely dispensable. Together, this work advances our knowledge of how the NCO versus CO outcome is determined during HR, expanding our understanding of how mitotic recombination is regulated.</p> / Dissertation

Thesis (Ph. D.)--Cornell University, August, 2006. / Vita. Includes bibliographical references (leaves 116-123).

Cdk7 (Cyclin-dependent kinase 7) is conserved from yeast to human and involved in multiple functions. Cdk7 acts as a CAK (Cdk activating kinase) in a trimeric complex with Cyclin H and Mat1. The CAK activity is required for the full activation of the Cdks that directly regulate the cell cycle transitions. In addition, Cdk7 is the kinase subunit of TFIIH, the general transcription/DNA repair factor IIH. TFIIH is required for the general transcription of messenger RNAs by RNA polymerase II and for the transcription-coupled nucleotide excision repair functions. As in other systems, Drosophila Cdk7 has multiple functions. In order to understand how different functions of Cdk7 are regulated, I performed genetic screens to identify the regulators or downstream factors of multiple functions of Cdk7. Several candidate dominant suppressors and enhancers were identified in these screens. One strong suppressor of cdk7, xpd, encodes another subunit of TFIIH. The genetic suppression by xpd attracted me to further characterize the biological significance of this interaction. I showed that Xpd does have a novel function in regulating CAK activity of cdk7 , it down-regulates mitotic CAK activity. Furthermore, I found that Xpd protein levels are cell cycle dependent, being down-regulated at the beginning of the mitosis. Based on these data, I propose a model that mitotic down-regulation of Xpd results in increased CAK activity, positively regulating mitotic progression. Simultaneously, this down-regulation can be expected to contribute to the mechanisms of mitotic silencing of basal transcription.

Thesis (Ph. D.)--Cornell University, May, 1998. / Vita. Includes bibliographical references (leaves 157-164).