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M.Sc. Zoology, University of Calcutta 2005
Thesis Advisor: Smita S. Patel, Ph.D
Graduate Program in Biochemistry & Molecular Biology
School of Public Health Building
2nd floor Conference Room
Thursday, October 27, 2011
Mitochondria are organelles of prokaryotic origin responsible for ATP synthesis in most eukaryotic cells. The human mitochondrial DNA (mtDNA) is a double stranded circular molecule of approximately 16 kb, present in 1000-10,000 copies per cell. The genome encodes 13 polypeptides involved in respiration, 2 rRNAs and 22 tRNAs. The proteins required for maintenance of the mitochondrial DNA (mtDNA) are encoded in the nucleus and transported to the mitochondria by special targeting sequences. Mutations in the mtDNA or the nuclear genes coding mtDNA maintenance proteins lead to mitochondrial genome instability culminating in several neuromuscular diseases in human. TWINKLE is a nucleus-encoded human mtDNA helicase which is similar in amino acid sequence to T7 gp4 helicase from T7 bacteriophage. It oligomerizes into a ring-shaped structure and unwinds DNA from 5-3 direction. Point mutations in TWINKLE are associated with heritable neuromuscular diseases characterized by deletions in the mtDNA. The general aim of this thesis is to biochemically characterize the mtDNA helicase TWINKLE and define its role in mtDNA transactions.
To this end, I purified recombinant TWINKLE from Escherichia coli without using any cofactors which allowed me to investigate the effects of the cofactors on its biochemical activities. By using protein-protein crosslinking, I have shown that E. coli expressed TWINKLE assembles into hexamers and higher oligomers, and addition of MgUTP stabilizes hexamers over higher oligomers. TWINKLE therefore makes a transition upon MgUTP binding from higher oligomers into hexamers which are active in loading on to the DNA substrate. Probing into the DNA unwinding activity of TWINKLE, I have observed that TWINKLE can load more efficiently on a single stranded DNA than on circular DNA, and it can use more than one type of NTP for DNA unwinding. I predict that in the cell a separate loader is required to load the helicase on the circular DNA.
In my in vitro assays, I have used short linear DNA substrates to follow the DNA unwinding or strand separation reaction, and have found that the efficiency of unwinding by TWINKLE is greatly enhanced when re-annealing of the displaced strands is prevented by including a heterologous single strand binding protein or a DNA trap. This happens because TWINKLE, though a helicase, has an antagonistic activity of annealing two complementary single-stranded (ss) DNA which interferes with unwinding. Although, TWINKLE binds both ssDNA and double stranded (ds) DNA with a high affinity as shown by my fluorescence anisotropy titrations, only ssDNA and not the dsDNA, competitively inhibits the annealing activity. These results indicate that TWINKLE has two types of DNA binding sites, and the ssDNA specific site catalyzes DNA annealing. The strand annealing activity of TWINKLE may play a role in recombination mediated replication initiation found in the mitochondria of mammalian brain and heart or in replication fork-regression during repair of damaged DNA replication forks. Supporting this hypothesis I have demonstrated that TWINKLE can mediate strand exchange reactions and can catalyze branch migration.
In the final part of the thesis, I have shown that TWINKLE needs a very specific 3 tail morphology in its unwinding substrate. This suggests that the 3 tail interacts with TWINKLE during unwinding likely via the external surface (N-terminal domain) of TWINKLE subunits. This interaction slows the helicase down when the 3 tail is single stranded in nature and stimulates it when the 3tail is double stranded in nature.
In summary, my thesis demonstrates that recombinant human mitochondrial helicase TWINKLE can be purified in an active form from E. coli. My in vitro studies of the purified TWINKLE has established multiple biochemical activites including hexamer formation, UTP hydrolysis, binding to ss and ds DNA, DNA strand separation, DNA annealing, and DNA strand-exchange reactions. These discoveries form the basis for defining the multi-faceted roles of TWINKLE in mtDNA metabolism.