Abstract
DNA helicases are motor proteins that couple the chemical energy of nucleoside triphosphate hydrolysis to the mechanical functions required for DNA unwinding. Studies of several helicases have identified strand-separating "pin" structures that are positioned to intercept incoming dsDNA and promote strand separation during helicase translocation. However, pin structures vary among helicases and it remains unclear whether they confer a conserved unwinding mechanism. Here, we tested the biochemical and cellular roles of a putative pin element within the Escherichia coli PriA DNA helicase. PriA orchestrates replication restart in bacteria by unwinding the lagging-strand arm of abandoned DNA replication forks and reloading the replicative helicase with the help of protein partners that combine with PriA to form what is referred to as a primosome complex. Using in vitro protein-DNA cross-linking, we localized the putative pin (a β-hairpin within a zinc-binding domain in PriA) near the ssDNA-dsDNA junction of the lagging strand in a PriA-DNA replication fork complex. Removal of residues at the tip of the β-hairpin eliminated PriA DNA unwinding, interaction with the primosome protein PriB, and cellular function. We isolated a spontaneous intragenic suppressor mutant of the priA β-hairpin deletion mutant in which 22 codons around the deletion site were duplicated. This suppressor variant and an Ala-substituted β-hairpin PriA variant displayed wildtype levels of DNA unwinding and PriB binding in vitro These results suggest essential but sequence nonspecific roles for the PriA pin element and coupling of PriA DNA unwinding to its interaction with PriB.
Highlights
DNA helicases are motor proteins that couple the chemical energy of nucleoside triphosphate hydrolysis to the mechanical functions required for DNA unwinding
To identify structures that could be important for PriA DNA unwinding, the Klebsiella pneumoniae and E. coli PriA helicase structures [13, 14] were superimposed with 3Ј-5Ј SF2 helicases for which structures with DNA substrates have been determined
As has been noted [13, 24, 34], the overlay showed that a -hairpin from the PriA CRR (residues 452– 462 in E. coli PriA (Fig. 1A)) was located at a similar position to pins in other helicases, implicating it as a putative strand-separation pin
Summary
DNA helicases are motor proteins that couple the chemical energy of nucleoside triphosphate hydrolysis to the mechanical functions required for DNA unwinding. Using in vitro protein–DNA cross-linking, we localized the putative pin (a -hairpin within a zinc-binding domain in PriA) near the ssDNA– dsDNA junction of the lagging strand in a PriA–DNA replication fork complex. An aromatic-rich loop/helicase motif IIa that interacts with DNA near the fork junction has been shown to be critical for coupling DNA binding to ATP hydrolysis and DNA unwinding [20] It is not clear, how this activity is linked to strand separation. The suppressor variant and an Ala-substituted -hairpin PriA variant displayed DNA unwinding and PriB interaction levels in vitro that were indistinguishable from WT PriA Together these results support a role for the PriA CRR -hairpin as a pin structure that is essential for DNA unwinding, protein partner (PriB) interaction, and cellular function. The sequence of the PriA pin was not critical to its function, suggesting that it functions as a simple physical wedge to aid DNA unwinding and that this wedge/ DNA interface is critical for subsequent PriB binding
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