title: Structural determinants for specific recognition by T4 endonuclease V.
abstract: DNA glycosylases catalyze the scission of the N-glycosyl bond linking either a damaged or mismatched base to the DNA sugar phosphate backbone. T4 endonuclease V is a glycosylase/apurinic (AP) lyase that is specific for UV light-induced cis-syn pyrimidine dimers. As a proposed transition state analog/inhibitor for glycosylases, a phosphoramidite derivative containing a pyrrolidine residue has been synthesized. The binding of endonuclease V to this duplex was analyzed by gel mobility shift assays and resulted in a single stable complex of reduced mobility and an apparent Kd of 17 nM. To assess the importance of the positive charge for specific binding, studies using other non-cleavable substrate analogs were performed. Wild type T4 endonuclease V shows an 8-fold decreased affinity for a tetrahydrofuran as compared with the pyrrolidine residue, demonstrating the significance of the positive charge for recognition. A 2-fold increase in binding affinity for a reduced AP site was observed. Similar assays using catalytically compromised mutants (E23Q and E23D) of endonuclease V demonstrate altered affinities for the pyrrolidine as well as tetrahydrofuran and reduced AP sites. This approach has provided insight into the structural mechanism by which specific lesions are targeted by the protein as well as the structural determinants of the DNA required for specific recognition by T4 endonuclease V.
authors.affiliation: Sealy Center for Molecular Science and Department of Human Biological Chemistry & Genetics, University of Texas Medical Branch, Galveston, Texas 77555-1071, USA.
authors.forename: A K
authors.initials: AK
authors.lastname: McCullough
authors.forename: O
authors.initials: O
authors.lastname: Schärer
authors.forename: G L
authors.initials: GL
authors.lastname: Verdine
authors.forename: R S
authors.initials: RS
authors.lastname: Lloyd
mesh_terms: D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics; D005663:Furans / Q000378:metabolism; D007700:Kinetics; D008190:Lyases / Q000037:antagonists & inhibitors; D016296:Mutagenesis; D011409:Propylene Glycols / Q000378:metabolism; D011487:Protein Conformation; D011759:Pyrrolidines / Q000378:metabolism; D013329:Structure-Activity Relationship; D014764:Viral Proteins
chemical_list: D005663:Furans; D011409:Propylene Glycols; D011759:Pyrrolidines; D014764:Viral Proteins; C018674:tetrahydrofuran; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer); D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; C032519:pyrrolidine