Investigating the molecular mechanisms of N-HELQ interaction with POLD3 and RPA1
Publication Date
July 27, 2026
Creator
Roberts, Alexander
Abstract
Human cells are at risk of DNA damage from a multitude of sources throughout their cell cycle, including during DNA replication. This can lead to DNA double-strand break (DSB) lesions in the genome, from which replication stress can arise, resulting in further DSBs and consequently genomic instability, which can in turn lead to genetic diseases, primarily cancer. Human cells have several DNA damage response pathways to counteract this, including homologous recombination, which comprises multiple sub-pathways. Of these, break
induced replication has the highest mutagenic potential. The DNA helicase HELQ acts to limit resultant mutagenic DNA synthesis through interaction of its N-terminal region N-HELQ with the single-stranded DNA binding protein RPA subunit RPA1 and DNA polymerase Pol δ subunit POLD3. This is of interest to the Bolt lab, with precise molecular mechanisms of these protein protein interactions (PPI) currently unknown. The work described in this thesis aimed to further the understanding of these mechanisms through identifying POLD3 and RPA1 amino acid residue sites necessary for the PPI.
In order to attempt this, the Bolt lab’s existing qualitative bimolecular fluorescence complementation (BiFC) assay time-course method was developed into an endpoint method to provide additional quantitative data. This was followed by an attempt to PE3b prime edit human U2OS osteosarcoma cells to investigate whether previous findings that HELQD142G/F143G is unable to displace ssDNA-bound RPA in vitro could be reproduced in vivo. Here it is shown using the developed BiFC assay method that N-HELQ interacts with multiple POLD3 intrinsically disordered protein regions (IDPRs) but is unable to interact with RPA1, hence favouring a native cell investigative approach. The subsequent prime editing was unsuccessful. Further research on this would need to utilise a far-red fluorescent protein in BiFC assays to allow quantitative time-course data, in conjunction with identifying POLD3 IDPR residues of interest in POLD3-N-HELQ PPI and hence generating mutant proteins for use in BiFC assays. Meanwhile, further prime editing would likely need to use an improved system such as PE7 to increase success probability. Together, these approaches can contribute to ultimately understanding the molecular mechanisms of HELQ interaction with Pol δ and RPA.
Item Type
ethesis
Thesis Type
MRes
Supervisors
Subjects (LC)
Associated Schools / Departments
UoN Repository URI
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Alexander Roberts MRes Thesis With Corrections.pdf
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