2026-09-20T13:51:02Zhttps://wakespace.lib.wfu.edu/server/oai/request

oai:null:10339/147602026-09-02T11:05:30Zcom_10339_14934col_10339_38132

Pence, Matthew 2009-06-04T16:08:39Z 2010-06-18T18:58:23Z 2009-06-04T16:08:39Z 2010-06-18T18:58:23Z 2009-06-04T16:08:39Z https://wakespace.lib.wfu.edu/handle/10339/14760 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. en_US Wake Forest University Translesion Synthesis Manganese Y family DNA polymerases Lesion Bypass of N2-ethylguanine by the Human Y Family DNA Polymerases Iota and Kappa Dissertation Release the entire work immediately for access worldwide. Wozniak, Daniel Perrino, Fred Hollis, Thomas Claiborne, Al Hantgan, Roy Biochemistry & Molecular Biology open.access