{"id":{"repo_id":"wfu","oai_identifier":"oai:wakespace.lib.wfu.edu:10339/14760"},"canonical_url":"https://search.dev.ndltd.org/etd/wfu/oai:wakespace.lib.wfu.edu:10339/14760","repository":{"repo_id":"wfu","name":"Wake Forest University","base_url":"https://wakespace.lib.wfu.edu/oai/request"},"display":{"title":"Lesion Bypass of N2-ethylguanine by the Human Y Family DNA Polymerases Iota and Kappa","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.","abstract_html":"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.","abstract_has_math":false,"creators":["Pence, Matthew"],"institution":"Wake Forest University","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2009,"date_issued":"2009-06-04T16:08:39Z","date_published":"2009-06-04T16:08:39Z","updated_at":"2026-07-27T22:01:01Z","subjects":["Translesion Synthesis"],"languages":["en_US"],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10339/14760","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Pence, Matthew"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2009-06-04T16:08:39Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2009-06-04T16:08:39Z"]},{"key":"dc:date.issued","label":"Date","values":["2009-06-04T16:08:39Z"]},{"key":"dc:publisher","label":"Institution","values":["Wake Forest University"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Translesion Synthesis"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en_US"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10339/14760"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:title","label":"Title","values":["Lesion Bypass of N2-ethylguanine by the Human Y Family DNA Polymerases Iota and Kappa"]}]}],"canonical_facts":{"dc:creator":["Pence, Matthew"],"dc:date.accessioned":["2009-06-04T16:08:39Z"],"dc:date.available":["2009-06-04T16:08:39Z"],"dc:date.issued":["2009-06-04T16:08:39Z"],"dc:description.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."],"dc:identifier.uri":["http://hdl.handle.net/10339/14760"],"dc:language.iso":["en_US"],"dc:publisher":["Wake Forest University"],"dc:subject":["Translesion Synthesis"],"dc:title":["Lesion Bypass of N2-ethylguanine by the Human Y Family DNA Polymerases Iota and Kappa"],"dc:type":["Dissertation"]},"updated_at":"2026-07-27T22:01:01Z"}