Functional hydrogen-bonding map of the minor groove binding tracks of six DNA polymerases.
Recent studies have identified amino acid side chains forming several hydrogen bonds in the DNA minor groove as potentially important in polymerase replication of DNA. Few studies have probed these interactions on the DNA itself. Using non-hydrogen-bonding nucleoside isosteres, we have now studied effects in both primer and template strands with several polymerases to investigate the general importance of these interactions. All six polymerases show differences in the H-bonding effects in the minor groove. Two broad classes of activity are seen, with a first group of DNA polymerases (KF(-), Taq, and HIV-RT) that efficiently extends nonpolar base pairs containing nucleoside Q (9-methyl-1H-imidazo[4,5-b]pyridine) but not the analogue Z (4-methylbenzimidazole), implicating a specific minor groove interaction at the first extension site. A second group of polymerases (Pol alpha, Pol beta, and T7(-)) fails to extend all non-H-bonding base pairs, indicating that these enzymes may need minor groove hydrogen bonds at both minor groove sites or that they are especially sensitive to noncanonical DNA structure or stability. All DNA polymerases examined use energetically important minor groove interactions to probe newly synthesized base pairs before extending them. The positions of these interactions vary among the enzymes, and only a subset of the interactions identified structurally appears to be functionally important. In addition, polymerases appear to be differently sensitive to small changes in base pair geometry.
Morales
J C
JC
Department of Chemistry, University of Rochester, Rochester, New York 14627, USA.
Kool
E T
ET
D000818:Animals; D017123:Bacteriophage T7 / Q000201:enzymology; D020029:Base Pairing; D001665:Binding Sites; D002417:Cattle; D004256:DNA Polymerase I / Q000737:chemistry; D019951:DNA Polymerase beta / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D054303:HIV Reverse Transcriptase / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D011489:Protein Denaturation; D019914:Taq Polymerase / Q000737:chemistry; D013698:Templates, Genetic; D013816:Thermodynamics
D017931:DNA Primers; D009692:Nucleic Acid Heteroduplexes; D019914:Taq Polymerase; C034970:bacteriophage T7 induced DNA polymerase; D054303:HIV Reverse Transcriptase; D004256:DNA Polymerase I; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase