2IMW

Mechanism of Template-Independent Nucleotide Incorporation Catalyzed by a Template-Dependent DNA Polymerase

Summary for 2IMW

• Entry DOI: 10.2210/pdb2imw/pdb
• Descriptor: 5'-D(*GP*GP*GP*GP*GP*AP*AP*GP*GP*AP*TP*TP*C)-3', 5'-D(*TP*AP*GP*AP*AP*TP*CP*CP*TP*TP*CP*CP*CP*CP*C)-3', DNA polymerase IV, ... (7 entities in total)
• Functional Keywords: blunt end dna y-family polymerase dna replication, transferase-dna complex, transferase/dna
• Biological source: Sulfolobus solfataricus
• Total number of polymer chains: 3
• Total formula weight: 49236.68

Authors

Ling, H.,Yang, W. (deposition date: 2006-10-05, release date: 2007-01-16, Last modification date: 2023-08-30)

Primary citation

Fiala, K.A.,Brown, J.A.,Ling, H.,Kshetry, A.K.,Zhang, J.,Taylor, J.S.,Yang, W.,Suo, Z.
Mechanism of Template-independent Nucleotide Incorporation Catalyzed by a Template-dependent DNA Polymerase.
J.Mol.Biol., 365:590-602, 2007

PubMed Abstract: 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.

PubMed: 17095011
DOI: 10.1016/j.jmb.2006.10.008

Experimental method

X-RAY DIFFRACTION (2.05 Å)