Mechanism of T7 Primase Selecting Active Priming Sites Among Genome

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Bacteriophage T7 primase initiates DNA replication by synthesizing primers.
• The known pentanucleotide recognition site (5'-(G/T)2GTC-3') is insufficient to fully explain primase activity.

Purpose of the Study:

• To investigate the role of flanking sequences in T7 primase binding to single-strand DNA (ssDNA).
• To elucidate the mechanism of T7 primase site selection for efficient genome replication.

Main Methods:

• Screening of approximately 26 T7 genome sequences for primase binding affinity and priming activity.
• Analysis of sequence features contributing to T7 primase-ssDNA interactions.

Main Results:

• The 10-nt sequence flanking the 3' end of the pentanucleotide site significantly influences T7 primase-ssDNA interactions.
• A specific dinucleotide within the 3' flank is critical for primase binding to ssDNA, potentially interacting with the zinc-binding domain.
• A multiple-site recognition model for T7 primase was proposed.

Conclusions:

• T7 primase utilizes extended ssDNA sequences, including a critical 3' dinucleotide, for precise priming site selection.
• This understanding provides insights into prokaryotic primase mechanisms and aids in developing novel inhibitors.