Browsing by Subject "helicase"

DNA replication is an intricate process within eukaryotic cells that must be precisely executed to preserve genetic information. This process begins at multiple start sites, or origins of replication, along each chromosome which are selected, licensed, and activated through cell-cycle regulated steps. Powerful reconstitution studies have identified the proteins involved in these processes, but they do not fully recapitulate the nuclear environment. Within the nucleus, the genome is organized in a chromatin structure consisting of DNA and all associated factors. At origins of replication, local chromatin contributes to origin identity and activation, but the precise chromatin dynamics that occur at these sites during helicase activation and initial DNA unwinding have not been fully explored. Additionally, how these steps are regulated to ensure genomic stability remain unstudied within the context of chromatin.

To address these questions, I have developed a conditional system that removes polymerase α function to capture helicase activation at replication origins in the budding yeast. Under restrictive conditions, these cells (cdc17-ts-FRB) do not initiate replication. When allowed to recover, replication appears to initiate outside origins, necessitating a delay in G2/M phase to repair unreplicated gaps at origins. To investigate origin chromatin and helicase movement prior to replication, I used MNase chromatin profiling alongside ChIP-seq for various replication factors. Chromatin in a 1 kb region around early, efficient replication origins is disrupted under restrictive conditions. The active helicase unwinds DNA out to 1 kb from these origins and is likely the source of the chromatin disruption. I next used the cdc17-ts-FRB conditional system to investigate the regulation of helicase progression in the absence of replication. I first tested whether the intra-S-phase checkpoint had a role in stalling the helicase 1 kb from the origin. Though removing checkpoint activation distributed helicase movement and chromatin disruption to late, inefficient origins, it did not alter the distance the helicase progressed from the origin. Instead, the helicase stalls as it leaves the AT-rich origin region and encounters sequences with higher GC content. These results provide in vivo support for the recently proposed “dead man’s switch” model for decreased helicase processivity when uncoupled from replication.

Helicase activation and origin unwinding are essential steps during DNA replication that expose ssDNA and thus have the potential to cause genomic instability. My studies have captured origin chromatin dynamics caused by an active helicase unwinding DNA, and have contributed evidence that the helicase may be intrinsically less processive in the absence of leading strand synthesis. These results may have implications for the mechanisms underlying human diseases involving polymerase α, and contribute to our growing understanding of how the eukaryotic cell preserves the integrity of the genome.

RecQ4, a member of the conserved RecQ family of helicases, is involved in replication and associated with several clinical syndromes. Although biologically important, the biochemistry of RecQ4 has remained elusive. We have expressed and purified Drosophila melanogaster RecQ4 from a baculovirus expression system. Biochemical characterization of the helicase, ATP hydrolysis, annealing, and binding activities of the enzyme has been performed, using native and non-native gel electrophoresis and thin layer chromatography, among other techniques. These reveal that RecQ4 is a 3' to 5' helicase that is stimulated by the presence of single-stranded DNA 3' of the duplex DNA region to be unwound. The enzyme is also capable of annealing complementary DNA strands, though this is inhibited by AMPPNP, a non-hydrolyzable analog of ATP. RecQ4 also forms a stable complex with single-stranded DNA in the presence of AMPPNP. We argue that the helicase activity of RecQ4 is important to the process of DNA replication. This leads to the conclusion that two helicases, RecQ4 and the Mcm2-7 complex, are involved in replication. The manner of their simultaneous involvement is not intuitive, and so models by which the two enzymes may cooperate are discussed.

Topoisomerase IIIα (Top3α) is an essential component of the double Holliday junction (dHJ) dissolvasome complex in metazoans. Previous work has shown that Top3α and Bloom's helicase (Blm) are able to convergently migrate the dHJ to create solely non-crossover products, thus preserving genomic integrity. However, many questions remain about the details of this process. Using a combination of biochemical and genetic tools, including dHJ substrate assays, gel electrophoresis, EMSA, pulldowns, fly crosses, and electron microscopy, this work expands our knowledge of the dissolution reaction. Tail mutants of Top3α were created and tested in a series of in vitro assays. Through these experiments, I discovered that the C-terminus of Top3α is important for binding Blm, interacting with DNA, conveying RPA stimulation, and in vivo functionality. I also observed that dissolution is an extremely processive reaction, with no accumulation of intermediates prior to product formation. When a non-specific topoisomerase was used (Top1, a type IB), accumulation of an intermediate was evident; however, contrary to predicted models, direct observation revealed that this intermediate is not a hemicatenane structure and still requires branch migration. Modifications were also made to the dHJ substrate creation method so that multiple types of HJ substrates could be produced efficiently.