title: How Y-Family DNA polymerase IV is more accurate than Dpo4 at dCTP insertion opposite an N2-dG adduct of benzo[a]pyrene.
abstract: To bypass DNA damage, cells have Y-Family DNA polymerases (DNAPs). One Y-Family-class includes DNAP κ and DNAP IV, which accurately insert dCTP opposite N(2)-dG adducts, including from the carcinogen benzo[a]pyrene (BP). Another class includes DNAP η and DNAP V, which insert accurately opposite UV-damage, but inaccurately opposite BP-N(2)-dG. To investigate structural differences between Y-Family-classes, regions are swapped between DNAP IV (a κ/IV-class-member) and Dpo4 (a η/V-class-member); the kinetic consequences are evaluated via primer-extension studies with a BP-N(2)-dG-containing template. Four key structural elements are revealed. (1) Y-Family DNAPs have discreet non-covalent contacts between their little finger-domain (LF-Domain) and their catalytic core-domain (CC-Domain), which we call "non-covalent bridges" (NCBs). Arg37 and Arg38 in DNAP IV's CC-Domain near the active site form a non-covalent bridge (AS-NCB) by interacting with Glu251 and Asp252, respectively, in DNAP IV's LF-Domain. Without these interactions dATP/dGTP/dTTP misinsertions increase. DNAP IV's AS-NCB suppresses misinsertions better than Dpo4's equivalent AS-NCB. (2) DNAP IV also suppresses dATP/dGTP/dTTP misinsertions via a second non-covalent bridge, which is ∼8Å from the active site (Distal-NCB). Dpo4 has no Distal-NCB, rendering it inferior at dATP/dGTP/dTTP suppression. (3) dCTP insertion is facilitated by the larger minor groove opening near the active site in DNAP IV versus Dpo4, which is sensible given that Watson/Crick-like [dCTP:BP-N(2)-dG] pairing requires the BP-moiety to be in the minor groove. (4) Compared to Dpo4, DNAP IV has a smaller major groove opening, which suppresses dGTP misinsertion, implying BP-N(2)-dG bulk in the major groove during Hoogsteen syn-adduct-dG:dGTP pairing. In summary, DNAP IV has a large minor groove opening to enhance dCTP insertion, a plugged major groove opening to suppress dGTP misinsertion, and two non-covalent bridges (near and distal to the active site) to suppress dATP/dGTP/dTTP misinsertions; collectively these four structural features enhance DNAP IV's dNTP insertion fidelity opposite a BP-N(2)-dG adduct compared to Dpo4.
authors.affiliation: Biology Department, Boston University, Boston, MA 02215, USA.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Sholder
authors.affiliation: Broad Institute, Cambridge, MA 02142, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: Creech
authors.affiliation: Biology Department, Boston University, Boston, MA 02215, USA. Electronic address: loechler@bu.edu.
authors.forename: Edward L
authors.initials: EL
authors.lastname: Loechler
mesh_terms: D019843:Archaeal Proteins / Q000378:metabolism; D001564:Benzo(a)pyrene / Q000633:toxicity; D001580:Benzopyrenes / Q000737:chemistry; D002274:Carcinogens, Environmental / Q000737:chemistry; D020134:Catalytic Domain / Q000235:genetics; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000737:chemistry*; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D009153:Mutagens / Q000633:toxicity; D048229:Sulfolobus solfataricus / Q000235:genetics; D013942:Thymine Nucleotides / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D001580:Benzopyrenes; D002274:Carcinogens, Environmental; D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D029968:Escherichia coli Proteins; D009153:Mutagens; D013942:Thymine Nucleotides; C478198:benzo(a)pyrene N2-dG adduct; C024107:2'-deoxycytidine 5'-triphosphate; D001564:Benzo(a)pyrene; C509838:Dpo4 protein, E coli; D019951:DNA Polymerase beta; D003849:Deoxyguanosine; C026600:2'-deoxyadenosine triphosphate; C024157:thymidine 5'-triphosphate
keywords: Adducts; Benzo[a]pyrene; Cancer; Lesion-bypass; Mutations; Y-Family DNA polymerases