Mechanism of template-independent nucleotide incorporation catalyzed by a template-dependent DNA polymerase.
Numerous template-dependent DNA polymerases are capable of catalyzing template-independent nucleotide additions onto blunt-end DNA. Such non-canonical activity has been hypothesized to increase the genomic hypermutability of retroviruses including human immunodeficiency viruses. Here, we employed pre-steady state kinetics and X-ray crystallography to establish a mechanism for blunt-end additions catalyzed by Sulfolobus solfataricus Dpo4. Our kinetic studies indicated that the first blunt-end dATP incorporation was 80-fold more efficient than the second, and among natural deoxynucleotides, dATP was the preferred substrate due to its stronger intrahelical base-stacking ability. Such base-stacking contributions are supported by the 41-fold higher ground-state binding affinity of a nucleotide analog, pyrene nucleoside 5'-triphosphate, which lacks hydrogen bonding ability but possesses four conjugated aromatic rings. A 2.05 A resolution structure of Dpo4*(blunt-end DNA)*ddATP revealed that the base and sugar of the incoming ddATP, respectively, stack against the 5'-base of the opposite strand and the 3'-base of the elongating strand. This unprecedented base-stacking pattern can be applied to subsequent blunt-end additions only if all incorporated dAMPs are extrahelical, leading to predominantly single non-templated dATP incorporation.
Fiala
Kevin A
KA
Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
Brown
Jessica A
JA
Ling
Hong
H
Kshetry
Ajay K
AK
Zhang
Jun
J
Taylor
John-Stephen
JS
Yang
Wei
W
Suo
Zucai
Z
D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D004247:DNA / Q000096:biosynthesis*; D019951:DNA Polymerase beta / Q000737:chemistry / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D007700:Kinetics; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011721:Pyrenes / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D013698:Templates, Genetic
D003838:Deoxyadenine Nucleotides; D009711:Nucleotides; D011721:Pyrenes; C119745:pyrene deoxynucleotide; D004247:DNA; D019951:DNA Polymerase beta; C026600:2'-deoxyadenosine triphosphate