Browsing by Subject "Deoxyribonucleic acid (DNA) replication"
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Item Characterization of XCdc7/Dbf4 and Cdc7/Drf1 in DNA replication initiation(Texas Tech University, 2006-12) Hirsch, Rachael; Coue, Martine; Schneider, Brandt; MacDonald, Clinton C.; Hutson, Jim; Whelly, Sandra M.Complete and accurate DNA replication is essential for proper cell division and thus the genetic integrity of all organisms. Several mechanisms regulate the process of DNA replication particularly during the initiation stage. Initiation must be carefully regulated to ensure that each segment of the DNA is replicated at the appropriate time, and only once per cell cycle. Cells achieve this by having a distinct stage that occurs prior to S-phase when replication origins become licensed for replication. At the onset of S-phase, replication forks are initiated only at these licensed origins. Licensing corresponds to the ordered assembly of multiprotein complexes celled pre-replication complexes (pre-RCs) onto the DNA at replication origins. These complexes are composed of Orc, Cdc6, Cdt1, and Mcm proteins. Once assembled the pre-RCs are activated at the appropriate time to trigger DNA replication. Two major kinases are known to participate in this activation, Cdk2/Cyclin E and Cdc7/Dbf4, though their mechanism of action is unknown. How these kinases regulate the initiation of replication is an important question underlying the regulation of the cell cycle and the maintenance of genomic integrity in all organisms. My study focuses on determining the role of the Cdc7 kinase in DNA replication initiation using an in vitro cell free system derived from Xenopus laevis eggs. Cdc7 kinase activity is reportedly dependent on a regulatory binding partner, called Dbf4. Members of our laboratory identified two potential Dbf4-related proteins in Xenopus laevis, XDbf4 and XDrf1. Since multiple Dbf4-related proteins within the same species had previously been reported in other organisms, I hypothesized that both of these proteins could interact with Xenopus Cdc7 to form active kinases that function during early embryonic DNA replication. In this study I show that both XDbf4 and XDrf1 physically interact with XCdc7 in interphase egg extracts to form two separate active kinase complexes. Furthermore I suggest that these two complexes are developmentally regulated indicating the possibility that they could function during different stages of early Xenopus development. Interestingly my data indicate that only the XCdc7/Drf1 complex is required for efficient early embryonic DNA replication. Furthermore, this complex seems to function specifically at the point of pre-RC activation, and not during pre-RC assembly. I also examined the mechanisms that could contribute to the regulation of the XCdc7 kinase. Cell-cycle mediated phosphorylation of the XCdc7 complex did not appear to affect its kinase activity. However, pre-phosphorylation of its XMcm2 substrate by Cdk did enhance its phosohorylation by XCdc7. Therefore, this could indicate a direct relationship between S-phase Cdks and the Cdc7 kinase. In addition to these studies, I also analyzed the potential involvement of the XCdc7 kinases in replication checkpoints that ensure that the cell cycle does not proceed without proper DNA replication. My studies indicated that these two XCdc7 kinases are not targeted by the checkpoint pathway. These studies are important in determining the manner in which the Cdc7 kinase contributes to the regulation of eukaryotic DNA replication.Item DNA superhelical density and CRP-mediated lac promoter activation(Texas Tech University, 1991-05) Lee, Hae KyungThe cyclic AMP receptor protein (CRP) functions in Escherichia coli in the regulation of several catabolite-sensitive operons. Cyclic AMP (cAMP) forms a complex with CRP which binds to specific DNA sites near the promoter of lactose operon {lacP) and activates the rate of its transcription by RNA polymerase. Mutant forms of the proteins (CRP*) that function independentiy of cAMP have been described. Studies were conducted to assess the response of the wild-type and mutant forms of CRP to changes in template DNA structure. To investigate the effect of varying template superhelical density (SHD) on CRP and CRP*-mediated lacP activity, in vitro transcription assays were performed. A lacP containing plasmid DNA, pJB3.5d/ac, was constructed and a series of topoisomers ranging from SHD = 0.000 to -0.098 were generated. The results of both full length transcripts and abortive initiation assays showed that CRP-mediated lacP activity was low at low template SHD and increased with increased template SHD under all conditions tested. Each mutant form of the protein was, however, unique in the range of DNA SHD providing maximal lacP stimulation. In addition, two mutant forms of CRP promoted RNA polymerase:/acP open complex formation in the absence of cAMP at half-times comparable to the CRPicAMP complex. The open complex formation half-times in reactions that contained wild-type CRPicAMP complex decreased with increasing SHD and are in good agreement with the published data. DNA:CRP binding properties of topoisomers were determined in experiments that utilize a product of in vivo recombination product, a small (451 base pairs) covalently closed circular DNA containing lacP region. The gel mobility shift assay on DNA:CRP:cAMP complex showed that in all DNA SHD examined, both wild-type and the mutant forms of CRP cause a retardation in migration rate, while in the absence of cAMP no difference in migration was observed. From the abortive initiation assay on 451 base pair (bp) DNA, it was found that lacP contained on the 451 bp circle does not show the same degree of cAMP dependence as lacP contained on a larger covalendy closed circle.