Thermodynamic, kinetic and structural basis for recognition and repair of abasic sites in DNA by apurinic/apyrimidinic endonuclease from human placenta.
X-ray analysis of enzyme-DNA interactions is very informative in revealing molecular contacts, but provides neither quantitative estimates of the relative importance of these contacts nor information on the relative contributions of specific and nonspecific interactions to the total affinity of enzymes for specific DNA. A stepwise increase in the ligand complexity approach is used to estimate the relative contributions of virtually every nucleotide unit of synthetic DNA containing abasic sites to its affinity for apurinic/apyrimidinic endonuclease (APE1) from human placenta. It was found that APE1 interacts with 9-10 nt units or base pairs of single-stranded and double-stranded ribooligonucleotides and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleotide phosphate groups. Such nonspecific interactions of APE1 with nearly every nucleotide within its DNA-binding cleft provides up to seven orders of magnitude (DeltaG degrees approximately -8.7 to -9.0 kcal/mol) of the enzyme affinity for any DNA substrate. In contrast, interactions with the abasic site together with other specific APE1-DNA interactions provide only one order of magnitude (DeltaG degrees approximately -1.1 to -1.5 kcal/mol) of the total affinity of APE1 for specific DNA. We conclude that the enzyme's specificity for abasic sites in DNA is mostly due to a great increase (six to seven orders of magnitude) in the reaction rate with specific DNA, with formation of the Michaelis complex contributing to the substrate preference only marginally.
Beloglazova
Natalia G
NG
Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Avenue, Novosibirsk 630090, Russia.
Kirpota
Oleg O
OO
Starostin
Konstantin V
KV
Ishchenko
Alexander A
AA
Yamkovoy
Vitaly I
VI
Zharkov
Dmitry O
DO
Douglas
Kenneth T
KT
Nevinsky
Georgy A
GA
D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008957:Models, Genetic; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism; D010920:Placenta / Q000201:enzymology; D013379:Substrate Specificity; D013816:Thermodynamics
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; D004247:DNA; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase