Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Biochemistry

Language

English (en)

Date of Award

1-1-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Sheila Stewart

Abstract

Coordination between DNA replication, DNA repair and cell-cycle progression ensures high fidelity DNA replication thus preventing mutations and DNA rearrangements. Interestingly, in addition to nuclear DNA stability, mitochondrial DNA: mtDNA) integrity is also essential for normal development. The current challenge resides in unraveling the different mechanisms that govern nuclear and mtDNA stability and to understand how these two separated genomes have evolved. This work focuses on delineating the biological functions of human Dna2: hDna2). Dna2 is a highly conserved helicase/nuclease that in yeast participates in DNA replication and Okazaki fragment maturation, DNA repair, and telomere maintenance. Immunofluorescence and biochemical fractionation studies demonstrated that in addition to its nuclear localization, hDna2 is also present inside the mitochondria where it colocalized with a subfraction of DNA-containing mitochondrial nucleoids in unperturbed cells. Upon the expression of disease-associated mutant forms of the mitochondrial Twinkle helicase, which induce DNA replication pausing/stalling, hDna2 accumulated within nucleoids suggesting that it participates in mtDNA replication/repair. In accordance with these observations, RNA interference-mediated depletion of hDna2 led to a decrease in mtDNA replication intermediates and inefficient repair of damaged mtDNA. I have also investigated the nuclear function of hDna2 and demonstrate that it participates in DNA replication. RNAi- mediated depletion of hDna2 led to nuclear genomic instability that is accompanied by the activation of the replication checkpoint kinase Chk1 in late S/G2 phase. Genetic rescue experiments revealed that both hDna2's nuclease and helicase activities are essential to maintain genomic stability, and suggest that these activities are coupled on long DNA flaps that arise during Okazaki fragments maturation. Furthermore, observations that hDna2 interacts with a member of the replisome, And-1, in a replication dependent manner, suggests that hDna2 is recruited to replication sites and actively participates in DNA replication. In accordance with biochemical and genetic models that predict that Dna2's activity is only required for a small percent of flaps that escape the activities of FEN1, hDna2 depletion did not result in slower maturation of newly synthesized DNA. In contrast, FEN1-depleted cells did result in slower maturation confirming that FEN1 is the main flap endonuclease that processes Okazaki fragments into ligable nicks. To establish whether hDna2 participates in DNA replication fork progression, we analyzed track length of replicating forks in vivo using micro-fluidic- assisted replication track analysis: maRTA). Surprisingly, we did not observe slowing of the replication fork upon hDna2 or FEN1 depletion suggesting that replication fork progression is insensitive to Okazaki fragment maturation. However, maRTA analysis revealed that origin firing events are reduced upon hDna2 depletion suggesting that hDna2 also participates in the firing of replication origins. In agreement with this hypothesis, chromatin immunoprecipitation: ChIP) analysis revealed that hDna2 specifically localizes to replication origins. Altogether, the work presented here demonstrates that hDna2 is a novel addition to the growing list of proteins that participate in both nuclear and mtDNA maintenance and further suggests that mechanisms of DNA replication/repair are conserved between both organelles. Furthermore, this work increases our understanding of the molecular mechanisms that ensure high fidelity replication and provides novel avenues in our quest to understand human diseases caused by mutations in DNA replication genes.

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Permanent URL: http://dx.doi.org/10.7936/K73F4MMP

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