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

During DNA replication, primases synthesize oligonucleotide primers on single-stranded template DNA, which are then extended by DNA polymerases to synthesize a complementary DNA strand. Primase RepB' of plasmid RSF1010 initiates DNA replication on two 40 nucleotide-long inverted repeats, termed ssiA and ssiB, within the oriV of RSF1010. RepB' consists of a catalytic domain and a helix bundle domain, which are connected by long α-helix 6 and an unstructured linker. Previous work has demonstrated that RepB' requires both domains for the initiation of dsDNA synthesis in DNA replication assays. However, the precise functions of these two domains in primer synthesis have been unknown. Here, we report that both domains of RepB' are required to synthesize a 10-12 nucleotide-long DNA primer, whereas the isolated domains are inactive. Mutational analysis of the catalytic domain indicates that the solvent-exposed W50 plays a critical role in resolving hairpin structures formed by ssiA and ssiB. Three structurally conserved aspartates (D77, D78, and D134) of RepB' catalyze the nucleotidyl transfer reaction. Mutations on the helix bundle domain are identified that either reduce the primer length to a dinucleotide (R285A) or abolish the primer synthesis (D238A), indicating that the helix bundle domain is required to form and extend the initial dinucleotide synthesized by the catalytic domain.