Translesion DNA Synthesis (TLS) is a mechanism that promotes DNA damage tolerance during DNA replication using an error-prone DNA polymerase complex. The complex is comprised of the ImuA, ImuB, and ImuC proteins that are found in approximately one-third of bacteria, including high priority antimicrobial resistant pathogens, such as Pseudomonas aeruginosa. Previous in vivo studies have shown that TLS increases beneficial bacterial mutations as the error-prone DNA polymerase, ImuC, lacks proof-reading activity. However, how ImuA and ImuB proteins contribute to the polymerase mechanism is unknown. Thus, the goal of this study is to characterize the TLS proteins in vitro to determine how ImuA and ImuB associate with ImuC to promote error- prone replication.
ImuA and ImuBNΔ34 were successfully purified for biochemical characterization from the homolog Myxococcus xanthus. Using size-exclusion chromatography coupled to multi-angle light scattering, both ImuA and ImuBNΔ34 are trimers in solution. Each protein also binds DNA independently as assessed by fluorescence polarization. Interestingly, both proteins bind ssDNA and a 3’ overhang substrate mimicking the DNA replication intermediate with the highest affinity. DNA binding assays further confirm these proteins can form a DNA-ImuA-ImuBNΔ34 complex. Using bacterial two-hybrid assays, the ImuA- ImuB interaction occurs in the C-terminal region of both proteins. Overall, these results suggest that ImuA and ImuB may recruit and stabilize ImuC on DNA for replication past damaged DNA, providing the first insights into the ImuA and ImuB molecular mechanism. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/27499 |
| Date | January 2022 |
| Creators | Tetenych, Andriana |
| Contributors | Andres, Sara, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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