Faithful replication of genomic information is crucial for maintenance of cellular function and integrity. DNA can adopt non-B-form structures in vivo, such as stable G-quadruplex DNA (G4 DNA). Evolutionarily retained G4 DNA is comprised of Hoogsteen hydrogen bonded guanines that form stable stacked tetrads. G4-forming sequences have been conserved in regulatory regions of the genome where they influence transcription and expression of important genes, including tumor suppressors and proto-oncogenes. Accurate and efficient G-quadruplex replication is critical to genomic maintenance and stability. Yet, the entire G-quadruplex structure represents a block to DNA synthesis by replicative polymerases (pols). Impaired replication of G-quadruplex DNA can propagate cellular stress and damage response mechanisms in cancer development and progression. Recent studies implicate human Y-family translesion synthesis (TLS) pols hRev1, hpol η, and hpol κ in successful G4 DNA replication. To better understand the mechanism of Y-family pol activity on G-quadruplex DNA, we examined the action of TLS pols hRev1, hpol η, and hpol κ on G4 substrates and compared them to the B-family hpol ϵ. We hypothesized that the unique structural and functional properties of the TLS pols would allow greater tolerance of G4 DNA than the more restrictive active sites of replicative pols like hpol ϵ. DNA binding experiments confirmed that the Y-family pols have increased affinity for G4 DNA substrates, while hpol ϵ has equal affinity for non-G4 and G4 DNA. Kinetic analyses revealed that hRev1 and hpol ? activities are impaired when inserting nucleotides opposite G4 DNA-guanines, while hpol η retained >25% catalytic efficiency on G4 substrates. Interestingly, hpol η and hpol κ exhibited increases in fidelity for nucleotide insertion on the G4 structure. Conversely, hpol ϵ activity was <5% and the fidelity of the replicative pol decreased on G4 DNA. Stopped-flow fluorescence assays revealed that hRev1 can disrupt G4 structures, and pol extension assays showed efficient replication of less stable G4 DNA by hpol η. Our studies support a model for replication of G4 DNA in which activities of individual replicative and TLS pols are partitioned to promote effective bypass of G-quadruplex structures. Furthermore, biochemical assays and experiments in cell culture revealed the importance of Y-family pol activity in the bypass/maintenance of G-quadruplex structures stabilized with G4 ligands, such as pyridostatin and porphyrins. This research provides fundamental insights into the mechanism(s) of G-quadruplex replication by specialized pols, which is essential for further studies exploring potential therapeutic options that target these structures and the proteins involved in their bypass.
|Advisor:||Eoff, Robert L.|
|Commitee:||Boerma, Marjan, Chambers, Timothy C., Miller, Grover P., Raney, Kevin D., Tackett, Alan J.|
|School:||University of Arkansas for Medical Sciences|
|Department:||Interdisciplinary Biomedical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 77/11(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Biochemistry|
|Keywords:||DNA replication, G-quadruplex, Kinetic partitioning, Rev1, Translesion synthesis, Y-family polymerase|
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