TY - JOUR
T1 - Biochemical evidence for the requirement of Hoogsteen base pairing for replication by human DNA polymerase ι
AU - Johnson, Robert E.
AU - Prakash, Louise
AU - Prakash, Satya
PY - 2005/7/26
Y1 - 2005/7/26
N2 - Because of the near geometric identity of Watson-Crick (W-C) G·C and A·T base pairs, a given DNA polymerase forms the four possible correct base pairs with nearly identical catalytic efficiencies. However, human DNA polymerase ι (Polι), a member of the Y family of DNA polymerases, exhibits a marked template specificity, being more efficient at incorporating the correct nucleotide opposite template purines than opposite pyrimidines. By using 7-deazaadenine and 7-deazaguanine as the templating residues, which disrupt Hoogsteen base pair formation, we show that, unlike the other DNA polymerases belonging to the A, B, or Y family, DNA synthesis by Polι is severely inhibited by these N7-modified bases. These observations provide biochemical evidence that, during normal DNA synthesis, template purines adopt a syn conformation in the Polι active site, enabling the formation of a Hoogsteen base pair with the incoming pyrimidine nucleotide. Additionally, mutational studies with Leu-62, which lies in close proximity to the templating residue in the Polι ternary complex, have indicated that both factors, steric constraints within the active site and the stability provided by the hydrogen bonds in the Hoogsteen base pair, contribute to the efficiency of correct nucleotide incorporation opposite template purines by Polι.
AB - Because of the near geometric identity of Watson-Crick (W-C) G·C and A·T base pairs, a given DNA polymerase forms the four possible correct base pairs with nearly identical catalytic efficiencies. However, human DNA polymerase ι (Polι), a member of the Y family of DNA polymerases, exhibits a marked template specificity, being more efficient at incorporating the correct nucleotide opposite template purines than opposite pyrimidines. By using 7-deazaadenine and 7-deazaguanine as the templating residues, which disrupt Hoogsteen base pair formation, we show that, unlike the other DNA polymerases belonging to the A, B, or Y family, DNA synthesis by Polι is severely inhibited by these N7-modified bases. These observations provide biochemical evidence that, during normal DNA synthesis, template purines adopt a syn conformation in the Polι active site, enabling the formation of a Hoogsteen base pair with the incoming pyrimidine nucleotide. Additionally, mutational studies with Leu-62, which lies in close proximity to the templating residue in the Polι ternary complex, have indicated that both factors, steric constraints within the active site and the stability provided by the hydrogen bonds in the Hoogsteen base pair, contribute to the efficiency of correct nucleotide incorporation opposite template purines by Polι.
KW - 7-deaza purines
KW - Syn and anti conformations
KW - Translesion DNA synthesis
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U2 - 10.1073/pnas.0503859102
DO - 10.1073/pnas.0503859102
M3 - Article
C2 - 16014707
AN - SCOPUS:23044439823
SN - 0027-8424
VL - 102
SP - 10466
EP - 10471
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 30
ER -