Mechanism of Template-independent Nucleotide Incorporation Catalyzed by a Template-dependent DNA Polymerase
- 作者：Kevin A. Fiala ; Jessica A. Brown ; Hong Ling ; Ajay K. Kshetry ; Jun Zhang ; John-Stephen Taylor ; Wei Yang ; Zucai Suo
- 关键词：pre-steady state kinetics ; X-ray crystal structure ; blunt-end addition ; Dpo4 ; pyrene nucleoside 5′ ; -triphosphate
- 刊名：Journal of Molecular Biology
- 出版年：2007
- 期刊代码：102_00222836
- 类别：imm
- 出版时间：19 January 2007
- 卷：365
- 期：3
- 页码：590-602
- 文件大小：1112 K
摘要
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 Å 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.