Document Detail

Zinc-binding domain of the bacteriophage T7 DNA primase modulates binding to the DNA template.
MedLine Citation:
PMID:  23024359     Owner:  NLM     Status:  MEDLINE    
The zinc-binding domain (ZBD) of prokaryotic DNA primases has been postulated to be crucial for recognition of specific sequences in the single-stranded DNA template. To determine the molecular basis for this role in recognition, we carried out homolog-scanning mutagenesis of the zinc-binding domain of DNA primase of bacteriophage T7 using a bacterial homolog from Geobacillus stearothermophilus. The ability of T7 DNA primase to catalyze template-directed oligoribonucleotide synthesis is eliminated by substitution of any five-amino acid residue-long segment within the ZBD. The most significant defect occurs upon substitution of a region (Pro-16 to Cys-20) spanning two cysteines that coordinate the zinc ion. The role of this region in primase function was further investigated by generating a protein library composed of multiple amino acid substitutions for Pro-16, Asp-18, and Asn-19 followed by genetic screening for functional proteins. Examination of proteins selected from the screening reveals no change in sequence-specific recognition. However, the more positively charged residues in the region facilitate DNA binding, leading to more efficient oligoribonucleotide synthesis on short templates. The results suggest that the zinc-binding mode alone is not responsible for sequence recognition, but rather its interaction with the RNA polymerase domain is critical for DNA binding and for sequence recognition. Consequently, any alteration in the ZBD that disturbs its conformation leads to loss of DNA-dependent oligoribonucleotide synthesis.
Seung-Joo Lee; Bin Zhu; Barak Akabayov; Charles C Richardson
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2012-09-28
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  287     ISSN:  1083-351X     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2012 Nov 
Date Detail:
Created Date:  2012-11-12     Completed Date:  2013-01-31     Revised Date:  2013-11-14    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  39030-40     Citation Subset:  IM    
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
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MeSH Terms
Amino Acid Sequence
Bacteriophage T7 / enzymology*
DNA / chemistry*
DNA Primase / chemistry*
DNA, Single-Stranded / genetics
Genetic Complementation Test
Molecular Sequence Data
Plasmids / metabolism
Protein Binding
Protein Conformation
Protein Structure, Tertiary
Recombinant Proteins / chemistry
Ribonucleotides / genetics
Surface Plasmon Resonance
Zinc / chemistry*
Grant Support
Reg. No./Substance:
0/DNA, Single-Stranded; 0/Recombinant Proteins; 0/Ribonucleotides; 7440-66-6/Zinc; 9007-49-2/DNA; EC 2.7.7.-/DNA Primase

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