3 research outputs found
Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase
We have used a stepwise increase in ligand complexity approach to estimate the relative contributions of the nucleotide units of DNA containing 7,8-dihydro-8-oxoguanine (oxoG) to its total affinity for human 8-oxoguanine DNA glycosylase (OGG1) and construct thermodynamic models of the enzyme interaction with cognate and non-cognate DNA. Non-specific OGG1 interactions with 10ā13ānt pairs within its DNA-binding cleft provides approximately 5 orders of magnitude of its affinity for DNA (ĪGĀ°āapproximately ā6.7ākcal/mol). The relative contribution of the oxoG unit of DNA (ĪGĀ° approximately ā3.3ākcal/mol) together with other specific interactions (ĪGĀ° approximately ā0.7ākcal/mol) provide approximately 3 orders of magnitude of the affinity. Formation of the Michaelis complex of OGG1 with the cognate DNA cannot account for the major part of the enzyme specificity, which lies in the kcat term instead; the rate increases by 6ā7 orders of magnitude for cognate DNA as compared with non-cognate one. The kcat values for substrates of different sequences correlate with the DNA twist, while the KM values correlate with ĪGĀ° of the DNA fragments surrounding the lesion (position from ā6 to +6). The functions for predicting the KM and kcat values for different sequences containing oxoG were found
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 enzymeāDNA 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 9ā10 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 (ĪGĀ° ā¼ ā8.7 to ā9.0 kcal/mol) of the enzyme affinity for any DNA substrate. In contrast, interactions with the abasic site together with other specific APE1āDNA interactions provide only one order of magnitude (ĪGĀ° ā¼ ā1.1 to ā1.5 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