Significance of nucleobase shape complementarity and hydrogen bonding in the formation and stability of the closed polymerase-DNA complex.
DNA polymerases insert a dNTP by a multistep mechanism that involves a conformational rearrangement from an open to a closed ternary complex, a process that positions the incoming dNTP in the proper orientation for phosphodiester bond formation. In this work, the importance and relative contribution of hydrogen-bonding interactions and the geometric shape of the base pair that forms during this process were studied using Escherichia coli DNA polymerase I (Klenow fragment, 3'-exonuclease deficient) and natural dNTPs or non-hydrogen-bonding dNTP analogues. Both the geometric fit of the incoming nucleotide and its ability to form Watson-Crick hydrogen bonds with the template were found to contribute to the stability of the closed ternary complex. Although the formation of a closed complex in the presence of a non-hydrogen-bonding nucleotide analogue could be detected by limited proteolysis analysis, a comparison of the stabilities of the ternary complexes indicated that hydrogen-bonding interactions between the incoming dNTP and the template increase the stability of the complex by 6-20-fold. Any deviation from the Watson-Crick base pair geometry was shown to have a destabilizing effect on the closed complex. This degree of destabilization varied from 3- to 730-fold and was found to be correlated with the size of the mismatched base pair. Finally, a stable closed complex is not formed in the presence of a ddNTP or rNTP. These results are discussed in relation to the steric exclusion model for the nucleotide insertion.
Dzantiev
L
L
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Alekseyev
Y O
YO
Morales
J C
JC
Kool
E T
ET
Romano
L J
LJ
D001482:Base Composition; D020029:Base Pairing; D004247:DNA / Q000737:chemistry*; D004256:DNA Polymerase I / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D003838:Deoxyadenine Nucleotides / Q000737:chemistry; D003854:Deoxyribonucleotides / Q000737:chemistry*; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry; D011721:Pyrenes / Q000737:chemistry; D012265:Ribonucleotides / Q000737:chemistry; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000737:chemistry; D014050:Toluene / Q000031:analogs & derivatives / Q000737:chemistry
C110637:2'-deoxy-1'-(2,4-difluorotol-5-yl)ribofuranose 5'-triphosphate; D017931:DNA Primers; D003838:Deoxyadenine Nucleotides; D003854:Deoxyribonucleotides; D009711:Nucleotides; D011721:Pyrenes; D012265:Ribonucleotides; D013942:Thymine Nucleotides; C119745:pyrene deoxynucleotide; D014050:Toluene; D004247:DNA; D004256:DNA Polymerase I; C026600:2'-deoxyadenosine triphosphate; C024157:thymidine 5'-triphosphate