TY - JOUR
T1 - T4 endonuclease V exists in solution as a monomer and binds to target sites as a monomer
AU - Latham, Katherine Atkins
AU - Rajendran, Surendran
AU - Carmical, J. Russ
AU - Lee, James C.
AU - Lloyd, R. Stephen
N1 - Funding Information:
We would like to thank J.-S. Taylor and Colin Smith (Washington University, St. Louis, MO) for preparing the CS 49mer substrate, F. Johnson, C. Iden, and A. Grollman (SUNY, Stony Brook) for synthesis of the THF 30met, and the UTMB Recombinant DNA Laboratory for synthesis of complementary oligonucleotides. We also thank Gary J. Latham for critical reading of this manuscript. This work was supported by NIH grants ES04091 and ES06676 and ACS FRA381 (R.S.L.) and grants H-0013 and H-1238 from the R.A. Welch Foundation (J.C.L). K.A.L. was supported by T32-ES07254.
PY - 1996/2/8
Y1 - 1996/2/8
N2 - 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.
AB - 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.
KW - Cyclobutane pyrimidine dimer
KW - Endonuclease V
KW - N-glycosylase
KW - Protein-DNA interaction
KW - Tetrahydrofuran
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U2 - 10.1016/0167-4838(95)00224-3
DO - 10.1016/0167-4838(95)00224-3
M3 - Article
C2 - 8597580
AN - SCOPUS:0343807317
SN - 0167-4838
VL - 1292
SP - 324
EP - 334
JO - Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
JF - Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
IS - 2
ER -