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Mechanism of DNA binding and sequence recognition by T7 DNA primase
Author(s) -
Lee SeungJoo,
Hamdan Samir M.,
Richardson Charles C.
Publication year - 2006
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.20.4.a512-c
Subject(s) - primase , dna , primer (cosmetics) , dna polymerase , biochemistry , dnag , biology , binding site , binding domain , dna replication , dna clamp , circular bacterial chromosome , chemistry , rna , gene , reverse transcriptase , organic chemistry
DNA primases catalyze the synthesis of oligoribonucleotides (primers) required for initiating DNA polymerization activity on the lagging strand. Prokaryotic primases are composed of two domains: a zinc‐binding domain (ZBD) that recognizes a specific sequence on DNA, and an RNA polymerase domain (RPD) that catalyzes the condensation of rNTPs. Previous structural and biochemical studies on bacteriophage T7 primase revealed that the RPD and ZBD are connected by a flexible region and that association of the two parts is essential for primer synthesis in a template‐dependent manner. However, little is known about the mechanism by which T7 primase recognizes a specific sequence on ssDNA. Using a combination of surface plasmon resonance and biochemical assays, we demonstrate that T7 primase preferentially binds to ssDNA containing a primase recognition sequence. Alteration in the ZBD results in loss of this specific DNA binding, while alteration in the RPD leads to a significant decrease in DNA binding affinity yet still retaining the specific sequence recognition. The binding of the primase to DNA and its sequence recognition is enhanced by the presence of ATP and CTP. We present a model in which binding of cognate NTPs to the active site induces conformational changes that facilitate contact of the ZBD with RPD (closed conformation) and consequently primer synthesis in a sequence dependent fashion. This work was supported by Public Health Service Grant GM 54397–4.