7-deazaadenine and purine

DNA damage that stalls replicative polymerases can be bypassed with the Y-family polymerases. These polymerases have more open active sites that can accommodate modified nucleotides. The lack of protein-DNA interactions that select for Watson-Crick base pairs correlate with the lowered fidelity of replication. Interstrand hydrogen bonds appear to play a larger role in dNTP selectivity. The mechanism by which purine-purine mispairs are formed and extended was examined with Solfolobus solfataricus DNA polymerase IV, a member of the RAD30A subfamily of the Y-family polymerases, as is pol eta. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 1-deaza- and 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. The time course of insertion of a single dNTP was examined with a polymerase concentration of 50 nM and a DNA concentration of 25 nM with various concentrations of dNTP. The time courses were fitted to a first-order equation, and the first-order rate constants were plotted against the dNTP concentration to produce k pol and K d (dNTP) values. A decrease in k pol/ K d (dNTP) associated with the deazapurine substitution would indicate that the position is involved in a crucial hydrogen bond. During correct base pair formation, the adenine to 1-deazaadenine substitution in both the incoming dNTP and template base resulted in a >1000-fold decrease in k pol/ K d (dNTP), indicating that interstrand hydrogen bonds are important in correcting base pair formation. During formation of purine-purine mispairs, the k pol/ K d (dNTP) values for the insertion of dATP and dGTP opposite 7-deazaadenine and 7-deazaguanine were decreased >10-fold with respect to those of the unmodified nucleotides. In addition, the rate of incorporation of 1-deaza-dATP opposite guanine was decreased 5-fold. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the anti conformation and the template base in the syn conformation. These results indicate that Dpo4 holds the incoming dNTP in the normal anti conformation while allowing the template nucleotide to change conformations to allow reaction to occur. This result may be functionally relevant in the replication of damaged DNA in that the polymerase may allow the template to adopt

Topics: Adenine; Archaeal Proteins; Base Pair Mismatch; Base Pairing; DNA Damage; DNA Polymerase beta; DNA Replication; Guanine; Hydrogen Bonding; Kinetics; Molecular Structure; Purines; Sulfolobus solfataricus

The mechanism by which purine-purine mispairs are formed and extended was examined with the high-fidelity Klenow fragment of Escherichia coli DNA polymerase I with the proofreading exonuclease activity inactivated. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. A decrease in rate associated with a 7-deazapurine substitution would suggest that the nucleotide is in a syn conformation in a Hoogsteen base pair with the opposite base. During mispair formation, the k(pol)/K(d) values for the insertion of dATP opposite A (dATP/A) as well as dATP/G and dGTP/G were decreased greater than 10-fold with the deazapurine in the dNTP. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the syn conformation and the template base in the anti conformation. During mispair extension, the only decrease in k(pol)/K(d) was associated with the G/G base pair in which 7-deazaguanine was in the template strand. These results as well as previous results [McCain et al. (2005) Biochemistry 44, 5647-5659] in which a hydrogen bond was found between the 3-position of guanine at the primer terminus and Arg668 during G/A and G/G mispair extension indicate that the conformation of the purine at the primer terminus is in the anti conformation during mispair extension. These results suggest that purine-purine mispairs are formed via a Hoogsteen geometry in which the dNTP is in the syn conformation and the template is in the anti conformation. During extension, however, the conformation of the primer terminus changes to an anti configuration while the template base may be in either the syn or anti conformations.

Topics: Adenine; Base Pair Mismatch; Base Sequence; DNA Polymerase I; DNA Replication; Escherichia coli; Guanine; Hydrogen Bonding; Kinetics; Molecular Sequence Data; Nucleic Acid Conformation; Purines; Structure-Activity Relationship

The effect of DNA bending on nucleobase electron transfer was investigated by studying the oxidation of double-stranded sequences containing seven repeats of the known bent sequence d(GGCA(1)A(2)A(3)A(4)A(5)A(6)C) where 7-deazaadenine (zA) was substituted at the A(3) position. Native gel electrophoresis was used to show that the sequence remained bent upon substitution of zA, which provides for oxidation of the sequence by Ru(bpy)(3)(3+) (bpy = 2,2'-bipyridine). The Ru(III) oxidant was generated by photolysis of Ru(bpy)(3)(2+) in the presence of ferricyanide, and the oxidation was visualized by high-resolution gel electrophoresis of the radiolabeled DNA sequence following base treatment. Cleavage of the DNA strand at the guanine residues and at the zA residues was observed. Comparison of the oxidation of zA in bent DNA versus the normal B form showed that hybridization of the B form sequence to its Watson-Crick complement produced a reduction in cleavage by a factor of 5.19 +/- 0.46 while hybridization of the bent sequence only reduced cleavage by a factor of 1.58 +/- 0.23. This result implies that the zA in the double-stranded, bent sequence is much more solvent-exposed than in normal B-form DNA. When the zA occurred in a B-form 5'-zA-G doublet, the reactivity was 6.63 +/- 0.10 times higher for the zA compared to the G. This implies an even greater effect of a 3'-guanine on the oxidation potential of zA than in the well-known 5'-GG doublet.

Topics: 2,2'-Dipyridyl; Adenine; DNA; Electrons; Indicators and Reagents; Models, Molecular; Nucleic Acid Conformation; Organometallic Compounds; Oxidation-Reduction; Purines; Solvents