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Binding of the DNA primase of bacteriophage T7 to its recognition sequence in DNA is modulated by the zinc‐binding domain
Author(s) -
Lee SeungJoo,
Zhu Bin,
Akabayov Barak,
Richardson Charles
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.648.1
Subject(s) - primase , dna , biology , gene , genetics , peptide sequence , primer (cosmetics) , recognition sequence , dna binding protein , mutagenesis , biochemistry , computational biology , mutation , chemistry , polymerase chain reaction , restriction enzyme , transcription factor , reverse transcriptase , organic chemistry
The zinc‐binding domains (ZBD) of prokaryotic DNA primases are critical for recognition of specific sequence in ssDNA at which primers are synthesized. ZBDs are structurally conserved in primases but they direct recognition of distinct trinucleotide sequences. In order to determine regions of the ZBD responsible for sequence‐specific recognition, we carried out homolog‐scanning mutagenesis on T7 DNA primase using a bacterial homolog from Bacillus stearothermophilus . Substitution of a five amino acid residue segment in the ZBD eliminates the ability of the primase to synthesize primers from a specific template sequence. The role of residues neighboring two cysteins (Cys17 and Cys20) in the primase was investigated by generating a library comprised of multiple amino acid substitutions at positions of Pro16, Asp18, and Asn19 followed by genetic screening for functional gene 4 protein. Examinations of the altered proteins reveal that substitutions at the three sites do not change the specific sequence recognized. However, the more positively charged residues in the region facilitates the better binding to a recognition site in a short template, leading to more efficient primer synthesis. Results from this study suggest that sophisticated architecture of ZBD determines recognition of specific sequence by DNA primase. This work was supported by Public Health Service Grant GM 54397.