Replication across template T/U by human DNA polymerase-iota.
Human DNA polymerase-iota (Poliota) incorporates correct nucleotides opposite template purines with a much higher efficiency and fidelity than opposite template pyrimidines. In fact, the fidelity opposite template T is so poor that Poliota inserts an incorrect dGTP approximately 10 times better than it inserts the correct dATP. We determine here how a template T/U is accommodated in the Poliota active site and why a G is incorporated more efficiently than an A. We show that in the absence of incoming dATP or dGTP (binary complex), template T/U exists in both syn and anti conformations, but in the presence of dATP or dGTP (ternary complexes), template T/U is predominantly in the anti conformation. We also show that dATP and dGTP insert differently opposite template T/U, and that the basis of selection of dGTP over dATP is a hydrogen bond between the N2 amino group of dGTP and Gln59 of Poliota.
Jain
Rinku
R
Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, NY 10029, USA.
Nair
Deepak T
DT
Johnson
Robert E
RE
Prakash
Louise
L
Prakash
Satya
S
Aggarwal
Aneel K
AK
D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D013698:Templates, Genetic; D013941:Thymine / Q000378:metabolism*; D014498:Uracil / Q000378:metabolism*; D014961:X-Ray Diffraction; D000094769:DNA Polymerase iota
D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D014498:Uracil; D004247:DNA; D005982:Glutathione Transferase; D004259:DNA-Directed DNA Polymerase; D013941:Thymine; D000094769:DNA Polymerase iota; C000722070:POLI protein, human