Helix Bundle Domain of Primase RepB' Is Required for Dinucleotide Formation and Extension

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• DNA replication relies on primases for primer synthesis and DNA polymerases for extension.
• Plasmid RSF1010 replication initiates via primase RepB' at inverted repeats ssiA and ssiB.
• RepB' possesses catalytic and helix bundle domains, both previously shown necessary for dsDNA synthesis.

Purpose of the Study:

• To elucidate the precise functions of RepB's catalytic and helix bundle domains in DNA primer synthesis.
• To investigate the roles of specific residues within these domains during initiation of DNA replication.

Main Methods:

• Site-directed mutagenesis of RepB' catalytic and helix bundle domains.
• In vitro DNA replication assays to assess primer synthesis and extension.
• Analysis of primer length and DNA synthesis initiation.

Main Results:

• Both RepB' domains are indispensable for synthesizing 10-12 nucleotide DNA primers; isolated domains are inactive.
• W50 in the catalytic domain is crucial for resolving ssiA/ssiB hairpin structures.
• Conserved aspartates (D77, D78, D134) catalyze nucleotidyl transfer.
• Helix bundle domain mutations yield dinucleotide primers (R285A) or abolish synthesis (D238A).

Conclusions:

• The catalytic domain initiates primer synthesis, while the helix bundle domain is vital for extending the initial dinucleotide.
• RepB' utilizes a multi-domain mechanism for efficient DNA primer synthesis and replication initiation.