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Lesion Bypass of N2-ethylguanine by the Human Y Family DNA Polymerases Iota and Kappa

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abstract
The purpose of this work is to test the hypothesis that the human Y family DNA polymerases have a biological function in the bypass of alkylation DNA damage. The lesion bypass properties of the Y family DNA polymerases ι and κ opposite the N2-ethylGua DNA adduct were studied using steady-state and rapid kinetics and structural analyses. Structures of two ternary complexes of DNA pol ι containing N2-ethylGua in the active site and with incoming dCTP or dTTP were solved. The structural data are evidence that the DNA pol ι utilizes Hoogsteen base-pairing as an efficient mechanism for nucleotide incorporation opposite N2-ethylGua. Comparisons between the N2-ethylGua containing structures of DNA pol ι and wild-type structures (PDB ID: 2ALZ and 2FLP) reveal that movements in a loop region of the Polymerase Associated Domain (PAD) allow accommodation of the adduct. The reorientation of the PAD loop defines the available space in the active site of DNA pol ι for binding of small N2-alkylGua lesions. Rates of nucleotide incorporation and extension by the DNA polymerases ι and κ opposite Gua and N2-ethylGua were determined using steady-state and single turnover kinetic assays with Mg2+ or Mn2+ as the activating metal. The efficiency of dCMP incorporation opposite N2-ethylGua by the DNA pol ι is increased ~2000-fold in the presence of Mn2+ compared to Mg2+. The increased efficiency is due to a decrease in the KM value for nucleotide binding and an increase in kcat. The pre-steady-state analysis of nucleotide incorporation by DNA pol κ opposite Gua and N2-ethylGua follows a biphasic kinetic model for nucleotide insertion. The data indicate that the burst amplitude is dependent on metal ion choice and concentration. The presence of N2-ethylGua results in reduced burst amplitudes for dCMP incorporation by DNA pol κ supporting Watson-Crick base-pairing during nucleotide incorporation opposite the adduct. Together the data indicate that the DNA polymerases ι and κ use distinct, yet efficient catalytic mechanisms for bypass of N2-ethylGua. The data support a role for the Y family DNA polymerases in the in vivo bypass of N2-ethylGua.
subject
Translesion Synthesis
Manganese
Y family DNA polymerases
contributor
Pence, Matthew (author)
Wozniak, Daniel (committee chair)
Perrino, Fred (committee member)
Hollis, Thomas (committee member)
Claiborne, Al (committee member)
Hantgan, Roy (committee member)
date
2009-06-04T16:08:39Z (accessioned)
2010-06-18T18:58:23Z (accessioned)
2009-06-04T16:08:39Z (available)
2010-06-18T18:58:23Z (available)
2009-06-04T16:08:39Z (issued)
degree
Biochemistry & Molecular Biology (discipline)
identifier
http://hdl.handle.net/10339/14760 (uri)
language
en_US (iso)
publisher
Wake Forest University
rights
Release the entire work immediately for access worldwide. (accessRights)
title
Lesion Bypass of N2-ethylguanine by the Human Y Family DNA Polymerases Iota and Kappa
type
Dissertation

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