Chemistry & Biochemistry Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2752
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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 PURIFICATION AND CHARACTERIZATION OF THE RECD PROTEIN-HOMOLOGUE FROM DEINOCOCCUS RADIODURANS(2004-12-06) Wang, Jianlei; Julin, Douglas A; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In many gram-negative bacteria, RecBCD enzyme is found to be responsible for double strand DNA break repair through homologous recombination. The AddAB enzyme, a RecBCD analog, is found in some gram-positive bacteria and functions in a similar way as RecBCD. A few bacteria appear to lack both RecBCD and AddAB enzymes entirely. One such organism is the bacterium Deinococcus radiodurans. This remarkable organism is able to survive in the presence of very high levels of radiation or DNA-damaging chemicals, levels that would overwhelm the DNA repair capacity of most other organisms. Interestingly, the D. radiodurans genome does have an open reading frame that would encode a protein that is homologous to the E. coli RecD protein. The amino acid sequence of this D. radiodurans RecD-like protein suggests that it is a helicase and therefore could function in some aspect of DNA repair, as does its E. coli homologue. However, the RecD protein of D. radiodurans must serve a different and novel function compared to the E. coli RecD protein. The D. radiodurans RecD protein can be expressed at high levels in E. coli and is readily purified by chromatography on a nickel column followed by single-stranded DNA-cellulose. The purified protein exhibits DNA-dependent ATPase and DNA helicase activities. The helicase activity requires at least a 10 nucleotide single strand overhang at the 5'-end of the double strand DNA substrate to start unwinding. The helicase assay shows that D. radiodurans RecD-like protein unwinds dsDNA substrates catalytically, but with low processivity, even with the help of single strand binding proteins (SSB) from either E. coli or D. radiodurans. These results show that D. radiodurans RecD-like protein is a DNA helicase that moves with 5'-3' polarity on single-stranded DNA. The E. coli RecD protein was shown recently to unwind dsDNA with the same 5'-3' polarity. The low processivity of the D. radiodurans RecD-like protein suggests that it may function in a complex with other proteins. The identity of these proteins is not known. We have also generated insertion mutations that are likely to disrupt all of the recD gene copies in the D. radiodurans genome after multiple generations growing in media with antibiotics. The in vivo effects of the insertion mutation, such as the growth curve and the sensitivity to UV radiation and DNA damaging chemicals, were studied.