T4 endonuclease V exists in solution as a monomer and binds to target sites as a monomer.
Endonuclease V, a N-glycosylase/lyase from T4 bacteriophage that initiates the repair of cyclobutane pyrimidine dimers in DNA, has been reported to form a monomer-dimer equilibrium in solution [Nickell and Lloyd (1991) Biochemistry 30, 8638], although the enzyme has only been crystallized in the absence of substrate as a monomer [Morikawa et al. (1992) Science 256, 523]. In this study, analytical gel filtration and sedimentation equilibrium techniques were used to rigorously characterize the association state of the enzyme in solution. In contrast to the previous report, at 100 mM KCl endonuclease V was found to exist predominantly as a monomer in solution by both of these techniques; no evidence for dimerization was seen. To characterize the oligomeric state of the enzyme at its target sites on DNA, the enzyme was bound to oligonucleotides containing a single site specific pyrimidine dimer or tetrahydrofuran residue. These complexes were analyzed by nondenaturing gel electrophoresis at various acrylamide concentrations in order to determine the molecular weights of the enzyme-DNA complexes. The results from these experiments demonstrate that endonuclease V binds to cyclobutane pyrimidine dimer and tetrahydrofuran site containing DNA as a monomer.
Latham
K A
KA
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555-1071, USA.
Rajendran
S
S
Carmical
J R
JR
Lee
J C
JC
Lloyd
R S
RS
D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011740:Pyrimidine Dimers; D012996:Solutions; D013816:Thermodynamics; D014461:Ultracentrifugation; D014764:Viral Proteins
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011740:Pyrimidine Dimers; D012996:Solutions; D014764:Viral Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer)