Theses and Dissertations from UMD
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Item Theoretical Studies of the Workings of Processive Molecular Motors(2017) Vu, Huong Thuy; Thirumalai, Devarajan; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Processive molecular motors, such as kinesins, myosins and helicases, take multiple discrete steps on linear polar tracks such as microtubules, filamentous actin, and DNA/RNA substrates. Insights into the mechanisms and functions of this important class of biological motors have been obtained through observations from single-molecule experiments and structural studies. Such information includes the distribution of n, the number of steps motors take before dissociating, and v, the motor velocity, in the presence and absence of an external resistive force from single molecule experiments; and different structures of different states of motors at different conditions. Based on those available data, this thesis focuses on using both analytical and computational theoretical tools to investigate the workings of processive motors. Two examples of processive motors considered here are kinesins that walk on microtubules while transporting vesicles, and helicases which translocate on DNA/RNA substrate while unwinding the helix substrate. New physical principles and predictions related to their motility emerge from the proposed theories. The most significant results reported in this thesis are: Exact and approximate equations for velocity distribution, P(v), and runlength distribution, P(n), have been derived. Application of the theory to kinesins shows that P(v) is non-Gaussian and bimodal at high resistive forces. This unexpected behavior is a consequence of the discrete spacing between the alpha/beta tubulins, the building blocks of microtubule. In the case of helicases, we demonstrate that P(v) of typical helicases T7 and T4 shows signatures of heterogeneity, inferred from large variations in the velocity from molecule to molecule. The theory is used to propose experiments in order to distinguish between different physical basis for heterogeneity. We generated a one-microsecond atomic simulation trajectory capturing the docking process of the neck-linker, a crucial element deemed to be important in the motility of Kinesin-1. The conformational change in the neck linker is important in the force generation in this type of motor. The simulations revealed new conformations of the neck-linker that have not been noted in previous structural studies of Kinesin-1, but which are demonstrated to be relevant to another superfamily member, Kinesin-5. By comparing the simulation results with currently available data, we suggest that the two superfamilies might actually share more similarities in the neck-linker docking process than previously thought.Item BIOCHEMICAL AND BIOLOGICAL CHARACTERIZATION OF THREE DNA REPAIR ENZYMES IN DEINOCOCCUS RADIODURANS(2009) Cao, Zheng; Julin, Douglas A; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Gram positive bacterium Deinococcus radiodurans is able to withstand acute doses of gamma rays that can cause hundreds of double-strand breaks per genome. In proposed double-stand break repair pathways, however, some important enzymes, such as helicases and nucleases in the initiation step, have not been clearly identified yet. Interestingly, the common bacterial helicase/nuclease complex RecBCD or AddAB, which functions to produce a 3' ssDNA tail in double-strand break repair initiation step in other bacteria, is not found in D. radiodurans. As part of efforts to identify helicases involved in double-strand break repair, the D. radiodurans HelIV (encoded by locus DR1572, the helD gene) was characterized with both in vivo and in vitro methods. The helD gene is predicted to encode a helicase superfamily I protein. The helD mutant is moderately sensitive to methyl methanesulfonate and hydrogen peroxide but it is not sensitive to gamma rays, UV and mitomycin C. In biochemical assays, the full length HelIV exhibited DNA unwinding activity with a 5'-3' polarity whereas the truncated HelIV without N-terminal region had no detectable helicase activity. RecJ is the exonuclease in the RecF pathway, which is suggested to function at the initiation step in DSB repair in the absence of RecBCD. In the in vivo study, the D. radiodurans recJ gene (encoded by locus DR1126) cannot be completely removed from the chromosome, indicating the essential role of RecJ in cell growth. The heterozygous mutant displayed growth defect and higher sensitivity to gamma rays, hydrogen peroxide and UV compared to wild type D. radiodurans, suggesting an important role in DNA repair. The RecJ expressed in E. coli system was insoluble but can be purified via denaturation-refolding, and the refolded RecJ showed 5'-3' exonuclease activity. D. radiodurans has no RecB and RecC proteins, but it has a homologue of the RecD protein. We tested whether the D. radiodurans RecD protein could form a complex or make transient interactions with other proteins to perform more complicated functions. The RecD conjugated protein affinity column was used to attempt to identify cellular binding partners.Item Biochemical characterization of the Minichromosome maintenance (MCM) helicase from Methanothermobacter thermautotrophicus(2009) Sakakibara, Nozomi; Julin, Douglas; Kelman, Zvi; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)DNA replication requires coordination of numerous proteins to duplicate genetic information in a precise and timely manner. One of the key players in replication is the replicative helicase that unwinds the duplex DNA to provide the single-stranded template for the DNA polymerases. Minichromosome maintenance (MCM) protein is the replicative helicase in archaea. This dissertation focuses on the MCM helicase from the euryarchaeon Methanothermobacter thermautotrophicus (Mth). Archaeal MCM proteins can be divided into two major parts, the N terminal and C terminal domains. The N terminal domain is essential for DNA binding and multimerization, while the C-terminus contains the catalytic domains. The objective of this dissertation is to elucidate the mechanism by which the N terminal domain communicates with the catalytic domain to facilitate helicase activity. To address this question, two approaches were taken. One approach identified conserved residues found in the N terminus and investigated their properties using various biochemical and biophysical methods. By analyzing several proteins with mutations in the conserved residues, a loop that is essential for MCM helicase activity was identified. The study suggests that the loop is involved in coupling the N-terminal DNA binding function and the catalytic activity of the AAA+ domain. Some other conserved residues, however, did not directly affect the MCM helicase activity but showed differences in biochemical properties suggesting that they may play a role in maintaining the structural integrity of the MCM helicase. Another approach determined the differences in thermal stability of the MCM protein in the presence of various cofactors and DNA substrates. The study shows that the protein has two unfolding transitions when ATP and the DNA are present, while non-hydrolyzable ATP results in one transition. This study suggests possible conformational changes arising from decoupling of two domains that occur during the ATP hydrolysis in the presence of DNA. Furthermore, both DNA binding function by the N terminal domain and ATP binding by the catalytic domain are essential for the change.Item Functions of the Tobacco mosaic virus helicase domain: regulating formation of the virus replication complex and altering the activity of a host-encoded transcription factor(2008-04-23) Wang, Xiao; Culver, James N; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tobacco mosaic virus (TMV)-encoded 126-kDa and 183-kDa replicases are multidomain and multifunctional proteins. The helicase domain shared by both replicases has been shown to perform multiple tasks during the virus life cycle. In vitro structural and functional analyses demonstrated that monomers and dimers of the TMV helicase domain were the active forms for ATP hydrolysis. However, self-interaction of the helicase polypeptides resulted in the formation of higher-order structures that likely serve as structural scaffolds for the assembly of virus replication complexes (VRCs). Mutagenesis studies of the TMV helicase motifs showed that conserved amino acid residues played important roles in protein ATPase and/or RNA binding activities. A close correlation between ATPase activity of the helicase domain and assembly of wild-type VRC-like vesicles by the 126-kDa replicase further suggests that ATPase activity of the TMV helicase domain may modulate proper VRC assembly. In addition to helicase self-interaction, a novel virus-host interaction involving ATAF2, a NAC domain transcription factor was identified. Members within the NAC domain family are involved in plant developmental processes and stress/defense responses. In this study, transgenic plants overexpressing ATAF2 showed a strong developmental phenotype. Inoculation of TMV in these transgenic plants resulted in reduced virus accumulations. Additionally, transcriptional induction of ATAF2 occurred in response to TMV infection and salicylic acid treatment. Combined, these results suggest that ATAF2 is involved in a host defense response. One interesting finding was that in susceptible hosts, virus-directed induction of ATAF2 and PR1, a well-defined pathogenesis-related (PR) marker gene for host defense system, occurred only in locally-infected but not in systemically-infected tissues. Dynamic changes in the expression of host defense genes suggest that viruses have evolved certain mechanisms to actively modulate host gene expression. Interaction between the TMV helicase domain and ATAF2 may provide one way to suppress the ATAF2-mediated host defense signaling pathway. Combined these studies investigated the importance of the TMV helicase domain in VRC formation and in manipulating the host defense system. The results confirmed the functional versatility of the TMV helicase domain in establishing a successful virus life cycle.Item EXPRESSING DEINOCOCCUS RADIODURANS RECD IN ESCHERICHIA COLI: PHENOTYPIC EFFECTS IN RECBCD(-) AND RECD(-) CELLS(2006-12-20) polansky, steven carl; Julin, Douglas A; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The RecD helicase is a member of the RecBCD complex, which is essential for repair of double-stranded breaks (DSBs) in DNA via homologous recombination in Escherichia coli. The microbe Deinococcus radiodurans is capable of fixing high amounts of DSBs, owing to an efficient repair system. However, Deinococcus does not contain any recB or recC genes; only a RecD-like helicase has been observed. In Escherichia coli strains mutant for the native RecD subunit, the D. radiodurans RecD cannot restore nuclease activity when infected with T4 gene2- bacteriophage. Cell viability tests and mitomycin C exposure of RecBC(+)D(-) strains show no adverse effects from the Dr RecD. A negative phenotype was encountered with strains lacking RecBCD and expressing D. radiodurans RecD protein. Microscopy studies of RecBCD(-) E. coli expressing the D. radiodurans RecD helicase show long cellular structures termed filaments. The Dr RecD protein may be binding to and disrupting the replication fork.