Multiple cleavage activities of endonuclease V from Thermotoga maritima: recognition and strand nicking mechanism.
Endonuclease V is a deoxyinosine 3'-endonuclease which initiates removal of inosine from damaged DNA. A thermostable endonuclease V from the hyperthermophilic bacterium Thermotoga maritima has been cloned and expressed in Escherichia coli. The DNA recognition and reaction mechanisms were probed with both double-stranded and single-stranded oligonucleotide substrates which contained inosine, abasic site (AP site), uracil, or mismatches. Gel mobility shift and kinetic analyses indicate that the enzyme remains bound to the cleaved inosine product. This slow product release may be required in vivo to ensure an orderly process of repairing deaminated DNA. When the enzyme is in excess, the primary nicked products experience a second nicking event on the complementary strand, leading to a double-stranded break. Cleavage at AP sites suggests that the enzyme may use a combination of base contacts and local distortion for recognition. The weak binding to uracil sites may preclude the enzyme from playing a significant role in repair of such sites, which may be occupied by uracil-specific DNA glycosylases. Analysis of cleavage patterns of all 12 natural mismatched base pairs suggests that purine bases are preferrentially cleaved, showing a general hierarchy of A = G > T > C. A model accounting for the recognition and strand nicking mechanism of endonuclease V is presented.
Huang
J
J
Department of Microbiology and Immunology, Hearst Microbiology Research Center and Strang Cancer Prevention Center, The Joan and Sanford I. Weill Medical College of Cornell University, 1300 York Avenue, Box 62, New York, New York 10021, USA.
Lu
J
J
Barany
F
F
Cao
W
W
D001080:Apurinic Acid / Q000378:metabolism; D020137:Base Pair Mismatch; D001483:Base Sequence; D001665:Binding Sites; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007288:Inosine / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D011119:Polynucleotides / Q000378:metabolism; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D014498:Uracil / Q000378:metabolism
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D011119:Polynucleotides; C001790:apyrimidinic acid; D001080:Apurinic Acid; D014498:Uracil; D007288:Inosine; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer)