Genetic and biochemical analysis of the Archaeal Helicase Hel308

Simmons, Emily (2019) Genetic and biochemical analysis of the Archaeal Helicase Hel308. MRes thesis, University of Nottingham.

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Abstract

Completion of semi-conservative DNA replication requires various DNA repair pathways to overcome both physical and chemical obstructions to the replisome. Hel308 is a DNA repair helicase important in the maintenance of genomic stability in archaea. It is particularly important in the repair of damage that blocks replication forks, with mutants exhibiting enhanced sensitivity to inter-strand cross-links. Hel308 is a Superfamily 2 DNA helicase, conserved throughout archaea, with homologues in metazoans, including humans. It is a monomeric, ssDNA-stimulated ATPase that translocates ssDNA in the 3’ to 5’ direction, and can unwind a variety of substrates in vitro, with a preference for forked DNA. It physically interacts with the ssDNA binding protein RPA and the recombinase Rad51. However, further functional details and the translocation mechanism of Hel308 remain poorly understood.

Here, analysis of Hel308 translocation was performed via the introduction of site-directed mutations into both the ratchet helix of domain IV, and a highly conserved motif within domain II (motif IVa) that appears to link the ATPase domains with the ratchet helix. This gave insight into the translocation mechanism of Hel308 and other helicases that possess these structural features. The importance of the ratchet helix in translocation, particularly its base-stacking interactions, was confirmed, and a potential auto-regulatory role of motif IVa was discovered. Insight was also gained into the role of Hel308 in homologous recombination.

Item Type: Thesis (University of Nottingham only) (MRes)
Supervisors: Bolt, Edward
Allers, Thorsten
Keywords: DNA repair helicase; Genomic stability; Translocation mechanism; Homologous recombination
Subjects: Q Science > QR Microbiology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 56053
Depositing User: Simmons, Emily
Date Deposited: 19 Jul 2019 04:40
Last Modified: 06 May 2020 14:31
URI: http://eprints.nottingham.ac.uk/id/eprint/56053

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