title: Dynamic characterization of a DNA repair enzyme: NMR studies of [methyl-13C]methionine-labeled DNA polymerase beta.
abstract: Crystallographic characterization of DNA polymerase beta (pol beta) has suggested that multiple-domain and subdomain motions occur during substrate binding and catalysis. NMR studies of [methyl-(13)C]methionine-labeled pol beta were conducted to characterize the structural and dynamic response to ligand binding. The enzyme contains seven methionine residues, one of which is at the amino terminus and is partially removed by the expression system. Three of the methyl resonances were readily assigned using site-directed mutants. Assignment of the resonances of Met155, Met158, and Met191 was more difficult due to the spatial proximity of these residues, so that assignments were based on NOESY-HSQC data and on the response to paramagnetic Co(2+) addition, as well as shift perturbations observed for the site-directed mutants. The response of the methyl resonances to substrate binding was evaluated by the serial addition of a template oligonucleotide, a downstream 5'-phosphorylated oligonucleotide, and a primer oligonucleotide to create a two-nucleotide-gapped DNA substrate. Addition of the single-stranded template DNA resulted in selective broadening of the methyl resonance of Met18 in the 8 kDa lyase domain, and this resonance then shifted and sharpened upon addition of a 5'-phosphate-terminated downstream complementary oligonucleotide. Conversion of the two-nucleotide-gapped DNA substrate to a single-nucleotide-gapped substrate by incorporation of ddCMP produced a small perturbation of the Met236 resonance, which makes contact with the primer strand in the crystal structure. The addition of a second equivalent of ddCTP to form the pol beta-DNA-ddCTP ternary complex resulted in significant shifts for the resonances corresponding to Met155, Met191, Met236, and Met282. The Met155 methyl resonance is severely broadened, while the Met191 and Met282 resonances exhibit significant but less extreme broadening. Since only Met236 makes contact with the substrate, the effects on Met155, Met236, and Met282 result from indirect conformational and dynamic perturbations. Previous crystallographic characterization of this abortive complex indicated that a polymerase subdomain or segment (alpha-helix N) repositions itself to form one face of the binding pocket for the nascent base pair. Met282 serves as a probe for motion in this segment. Addition of Mg(2+)-dATP to pol beta in the absence of DNA produced qualitatively similar but much smaller effects on Met191 and Met155, but did not strongly perturb Met282, leading to the conclusion that Mg(2+)-dATP alone is insufficient to produce the large conformational changes that are observed in the abortive complex involving the gapped DNA with a blocked primer and ddNTP. Thus, the NMR data indicate that the nucleotide-DNA interaction appears to be essential for conformational activation.
authors.affiliation: Laboratory of Structural Biology, NIEHS, NIH, Research Triangle Park, North Carolina 27709, USA.
authors.forename: Bidisha
authors.initials: B
authors.lastname: Bose-Basu
authors.forename: Eugene F
authors.initials: EF
authors.lastname: DeRose
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Kirby
authors.forename: Geoffrey A
authors.initials: GA
authors.lastname: Mueller
authors.forename: William A
authors.initials: WA
authors.lastname: Beard
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson
authors.forename: Robert E
authors.initials: RE
authors.lastname: London
mesh_terms: D000818:Animals; D002247:Carbon Isotopes; D019951:DNA Polymerase beta / Q000737:chemistry*; D045643:DNA Repair Enzymes / Q000737:chemistry*; D003845:Deoxycytosine Nucleotides / Q000737:chemistry / Q000494:pharmacology; D015394:Molecular Structure; D009038:Motion; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D051381:Rats
chemical_list: D002247:Carbon Isotopes; D003845:Deoxycytosine Nucleotides; D009841:Oligonucleotides; D019951:DNA Polymerase beta; D045643:DNA Repair Enzymes