title: Single Qdot-labeled glycosylase molecules use a wedge amino acid to probe for lesions while scanning along DNA.
abstract: Within the base excision repair (BER) pathway, the DNA N-glycosylases are responsible for locating and removing the majority of oxidative base damages. Endonuclease III (Nth), formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) are members of two glycosylase families: the helix-hairpin-helix (HhH) superfamily and the Fpg/Nei family. The search mechanisms employed by these two families of glycosylases were examined using a single molecule assay to image quantum dot (Qdot)-labeled glycosylases interacting with YOYO-1 stained λ-DNA molecules suspended between 5 µm silica beads. The HhH and Fpg/Nei families were found to have a similar diffusive search mechanism described as a continuum of motion, in keeping with rotational diffusion along the DNA molecule ranging from slow, sub-diffusive to faster, unrestricted diffusion. The search mechanism for an Fpg variant, F111A, lacking a phenylalanine wedge residue no longer displayed slow, sub-diffusive motion compared to wild type, suggesting that Fpg base interrogation may be accomplished by Phe(111) insertion.
authors.affiliation: The Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405, USA.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Dunn
authors.forename: Neil M
authors.initials: NM
authors.lastname: Kad
authors.forename: Shane R
authors.initials: SR
authors.lastname: Nelson
authors.forename: David M
authors.initials: DM
authors.lastname: Warshaw
authors.forename: Susan S
authors.initials: SS
authors.lastname: Wallace
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry* / Q000378:metabolism; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000737:chemistry* / Q000378:metabolism; D004058:Diffusion; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009994:Osmolar Concentration; D010649:Phenylalanine / Q000737:chemistry; D045663:Quantum Dots
chemical_list: D029968:Escherichia coli Proteins; D010649:Phenylalanine; D004247:DNA; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; C468346:Nei protein, E coli; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli