Interaction of Endonuclease Eco RI with Short Specific and Nonspecific Oligonucleotides

Abstract

SummaryThe interaction of EcoRI with different oligodeoxyribonucleotides (ODNs) was analyzed using the method of the slow step-by-

Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase

“…The present study shows that the recognition of non-cognate DNA by OGG1 is similar to its recognition by DNA polymerases (7,8,45), UNG and Fpg DNA glycosylases (10–12), APEX1 (13), topoisomerase I (15,16), Eco RI (14), HIV integrase (17), RecA protein (6), DNA ligase I and RNA helicase p68 (DDX5) (3) in terms of additivity of Gibbs free energies. Usually, the footprint of 30–40 kDa enzymes, including OGG1, covers one helix turn or 10 base pairs of DNA, and their active sites bind one singled out cognate or non-cognate nucleotide unit with a relatively high affinity.…”

“…In contrast to DNA polymerases, human uracil–DNA glycosylase (UNG), AP endonuclease (APEX1), and topoisomerase I (3,7,8,10,13,15,16) but similarly to E. coli Fpg and Eco RI endonuclease (11,12,14), the affinity of OGG1 for non-cognate d(pC) n , d(pT) n and d(pA) n did not depend on the relative hydrophobicity of their bases (Figure 3). The affinity of a 23-mer mixed-sequence ODN G11 (20 µM) was the same as that for all homo-d(pN) 23 (17–20 µM; Table 1).…”

“…Usually, the footprint of 30–40 kDa enzymes, including OGG1, covers one helix turn or 10 base pairs of DNA, and their active sites bind one singled out cognate or non-cognate nucleotide unit with a relatively high affinity. In contrast to many enzymes but similarly to UNG and EcoR I (10,14), OGG1 forms efficient contacts only with the internucleotide phosphate groups of non-cognate ds DNA. The f factor (1.78) for OGG1, reflecting its interaction with one internucleotide phosphate ( K d characterizing the enzyme interaction with one nucleotide unit is 1/ f  = 1/1.78 = 0.56 M), is comparable with f factors for other enzymes: 1.35 for human UNG; 1.45 for human APEX1; 1.52 for Klenow fragment of DNA polymerase I from E. coli ; 1.54 for E. coli Fpg; 1.61 for human RNA helicase p68; 1.3–2.0 for HIV-1 integrase; 2.0 for Eco RI, and 2.14 for human DNA ligase I.…”

“…containing specific features recognized by the particular protein, such as a recognition sequence of a restriction endonuclease, or a DNA lesion for DNA repair proteins) or non-cognate (general DNA containing no such specific features). Using the SILC approach, we have studied a number of DNA-dependent enzymes including those not specific for DNA structure or sequence such as Escherichia coli RecA (6), specific for DNA structure but not for sequence such as DNA polymerases of prokaryotes, eukaryotes, viruses, and archaea (3,7,8) and human DNA ligase I (3), specific for DNA damage such as human uracil DNA glycosylase (UNG) (9,10), E. coli 8-oxoguanine DNA glycosylase (Fpg) (11,12), and human apurinic/apyrimidinic (AP) endonuclease (APEX1) (13), and specific for DNA sequence such as Eco RI restriction endonuclease (14), human topoisomerase I (15,16), and HIV integrase (17). We have shown that all these enzymes recognize DNA by forming multiple additive contacts with all DNA units covered by the protein globule (7–20 depending on the enzyme), and that the total interaction is a combination of weak electrostatic and hydrophobic and/or van der Waals interactions of the enzyme with the individual structural elements of DNA, which can be described by the following equation: where K d [( P i )] is the dissociation constant ( K d ) for the minimal ortho phosphate ligand, e is the electrostatic factor reflecting the increase in enzyme affinity due to the interaction with one internucleoside phosphate group, h are hydrophobic factors for A, C, G and T nucleotide bases, the numbers of which in d(pN) n are a , c , g and t , respectively.…”

Structural, thermodynamic, and kinetic basis for the activities of some nucleic acid repair enzymes

“…Later, sequence‐independent human DNA ligase and RNA helicase (Nevinsky, 2003), and several sequence‐specific enzymes, such as Eco RI restriction endonuclease (Kolocheva et al ., 2001), human topoisomerase I (Figure 1B) (Bugreev et al ., 2003a; Bugreev et al ., 2003b, 2003c), and HIV integrase (Bugreev et al ., 2003d) were studied using the SILC approach. It was shown that all these enzymes recognize DNA by forming multiple additive contacts with all DNA elements covered by the protein globule ( n = 7–20 depending on the enzyme), and that the interaction of sequence‐independent enzymes with any DNA and sequence‐specific enzymes with nonspecific DNA can be described by the same Equation ( 1 ) (Bugreev and Nevinsky, 1999; Kolocheva et al ., 2001; Bugreev et al ., 2003a, 2003b, 2003c, 2003d; Nevinsky, 2003; Nevinskii, 2004). Only the values of e and h N factors and K d of orthophosphate change from one enzyme to another and from ss to ds DNA (Nevinsky, 2003; Nevinskii, 2004) (Tables 1 and 2).…”