The Escherichia Coli PriA Helicase Specifically Recognizes Gapped DNA Substrates

Abstract

The primosome is a multi-protein-DNA complex that catalyzes the priming of the DNA during the replication process. In Escherichia coli, PriA helicase plays a fundamental role in the initiation of the ordered assembly of the primosome. PriA is involved in recombination and repair processes being a major factor that initiates the restart of the stalled replication fork at the damaged DNA sites. This happens, presumably, through the recognition of the damaged DNA site structure though the nature of this recognition process is unknown. Here we present quantitative studies of the ssDNA gap recognition by the PriA helicase and the effect of the nucleotide cofactors on the recognition process. The data indicate a surprisingly low minimum total site size of the enzyme in the gap complex, which is ∼7 nucleotides or bp as compared with the site size of ∼20 nucleotides of the enzyme-ssDNA complex. The low stoichiometry indicates that the helicase exclusively engages the strong DNA-binding subsite in the gap complex and assumes a very different orientation, compared with the complex with the ssDNA. The PriA helicase binds the ssDNA gaps with 4-5 nucleotides with the highest affinity without engaging in cooperative interactions with the enzyme molecules associated with the surrounding dsDNA. Binding of ADP to strong and weak nucleotide-binding sites of the enzyme profoundly affects the affinity and stoichiometry of the helicase-gapped DNA complex. These observations are of fundamental importance for understanding of the enzyme mechanisms in both replication and recombination processes.