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Evidence for the kinetic partitioning of polymerase activity on G-quadruplex DNA.

ABSTRACT: We have investigated the action of the human DNA polymerase ? (hpol ?) and ? (hpol ?) catalytic cores on G-quadruplex (G4) DNA substrates derived from the promoter of the c-MYC proto-oncogene. The translesion enzyme hpol ? exhibits a 6.2-fold preference for binding to G4 DNA over non-G4 DNA, while hpol ? binds both G4 and non-G4 substrates with nearly equal affinity. Kinetic analysis of single-nucleotide insertion by hpol ? reveals that it is able to maintain >25% activity on G4 substrates compared to non-G4 DNA substrates, even when the primer template junction is positioned directly adjacent to G22 (the first tetrad-associated guanine in the c-MYC G4 motif). Surprisingly, hpol ? fidelity increases ~15-fold when copying G22. By way of comparison, hpol ? retains ~4% activity and has a 33-fold decrease in fidelity when copying G22. The fidelity of hpol ? is ~100-fold greater than that of hpol ? when comparing the misinsertion frequencies of the two enzymes opposite a tetrad-associated guanine. The kinetic differences observed for the B- and Y-family pols on G4 DNA support a model in which a simple kinetic switch between replicative and TLS pols could help govern fork progress during G4 DNA replication.

SUBMITTER: Eddy S

PROVIDER: S-EPMC4682564 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Evidence for the kinetic partitioning of polymerase activity on G-quadruplex DNA.

Eddy Sarah S Maddukuri Leena L Ketkar Amit A Zafar Maroof K MK Henninger Erin E EE Pursell Zachary F ZF Eoff Robert L RL

Biochemistry 20150511 20

We have investigated the action of the human DNA polymerase ε (hpol ε) and η (hpol η) catalytic cores on G-quadruplex (G4) DNA substrates derived from the promoter of the c-MYC proto-oncogene. The translesion enzyme hpol η exhibits a 6.2-fold preference for binding to G4 DNA over non-G4 DNA, while hpol ε binds both G4 and non-G4 substrates with nearly equal affinity. Kinetic analysis of single-nucleotide insertion by hpol η reveals that it is able to maintain >25% activity on G4 substrates compa ...[more]

PMID: 25903680

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Project description:Telomeres are specialized chromatin structures found at the end of chromosomes and are crucial to the maintenance of eukaryotic genome stability. Human telomere DNA is comprised of the repeating sequence (T2AG3)n, which is predominantly double-stranded but terminates with a 3' single-stranded tail. The guanine-rich tail can fold into secondary structures known as a G-quadruplexes (GQs) that may exist as a polymorphic mixture of anti-parallel, parallel, and several hybrid topological isomers. Using single-molecule Förster resonance energy transfer (smFRET), we have reconstructed distributions of telomere DNA GQ conformations generated by an in situ refolding protocol commonly employed in single-molecule studies of GQ structure, or using a slow cooling DNA annealing protocol typically used in the preparation of GQ samples for ensemble biophysical analyses. We find the choice of GQ folding protocol has a marked impact on the observed distributions of DNA conformations under otherwise identical buffer conditions. A detailed analysis of the kinetics of GQ folding over timescales ranging from minutes to hours revealed the distribution of GQ structures generated by in situ refolding gradually equilibrates to resemble the distribution generated by the slow cooling DNA annealing protocol. Interestingly, conditions of low ionic strength, which promote transient GQ unfolding, permit the fraction of folded DNA molecules to partition into a distribution that more closely approximates the thermodynamic folding equilibrium. Our results are consistent with a model in which kinetic partitioning occurs during in situ folding at room temperature in the presence of K(+) ions, producing a long-lived non-equilibrium distribution of GQ structures in which the parallel conformation predominates on the timescale of minutes. These results suggest that telomere DNA GQ folding kinetics, and not just thermodynamic stability, likely contributes to the physiological ensemble GQ structures.

Dataset Information

Evidence for the kinetic partitioning of polymerase activity on G-quadruplex DNA.

Publications

Evidence for the kinetic partitioning of polymerase activity on G-quadruplex DNA.

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