On the role of translesion synthesis polymerase Rev1 in homologous recombination and DNA damage tolerance
Abstract: DNA is frequently damaged by exogenous and endogenous agents. DNA
lesions are particularly dangerous during DNA replication as replicative polymerases
associated with the replication fork cannot efficiently utilize modified bases as
templates. As a result, the replication fork can stall as it encounters DNA lesions, and
ultimately collapse, if the stall persist. Collapsed forks resul... read moret in DNA double strand
breaks (DSBs), which can lead to mutations, translocations, and cell death. Fortunately,
cells have developed DNA damage tolerance (DDT) to prevent fork collapse, and DSB repair
pathways to repair DSBs if collapse occurs. DSBs can also occur outside of replication and
can be repaired through homologous recombination (HR) or end-joining. Specialized
translesion synthesis (TLS) polymerases are frequently employed in these pathways. A key
TLS polymerase, Rev1 is utilized during both DDT and DSB break repair; however, its role in
both pathways is not completely understood. We used Drosophila melanogaster to investigate
the role of Rev1 in both DDT and DSB repair. To do so, we created a complete rev1 knockout
and determined its ability to carry out HR repair of DSBs with differing synthesis
requirements. We find that Rev1 antagonizes HR regardless of the size of the DSB and the
amount of synthesis necessary for repair. Depending on type of break, the inhibition of HR
by Rev1, however, may occur through differing mechanisms. We also characterized DDT in
Drosophila, and find that Rev1 is essential for DDT following DNA alkylation damage. rev1
mutants were hypersensitive to the DNA alkylating agent, methyl methansulfonate (MMS). The
sensitivity of rev1 was equivalent to Drosophila lacking both DDT pathways, TLS, and the
recombination mediated template switch. We propose that Rev1 may promote both TLS and
template switching. To determine other genes within template switching, we conducted a
template switch specific ethyl methansulfonate (EMS) mutagenesis screen, and found 17
candidate isolates. Lastly, we investigated how the DNA sequence surrounding a DSB can
influence the repair junction formed by the alternative end-joining pathway,
synthesis-dependent microhomology-mediated end-joining (SD- MMEJ). Together these studies
begin to elucidate the enigmatic role of Rev1 in HR and DDT. In addition, we begin to
understand how the sequence surrounding a DSB affects SD-MMEJ.
Thesis (Ph.D.)--Tufts University, 2017.
Submitted to the Dept. of Biology.
Advisor: Mitch McVey.
Committee: Sergei Mirkin, Stephen Fuchs, Erik Dopman, and Julian Sale.
Keywords: Biology, Genetics, and Molecular biology.read less