An aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase

Abstract

Helicases couple ATP hydrolysis to nucleic acid binding and unwinding via molecular mechanisms that remain poorly defined for most enzyme subfamilies within the superfamily 2 (SF2) helicase group. A crystal structure of the PriA SF2 DNA helicase, which governs restart of prematurely terminated replication processes in bacteria, revealed the presence of an aromatic-rich loop (ARL) on the presumptive DNA-binding surface of the enzyme. The position and sequence of the ARL was similar to loops known to couple ATP hydrolysis with DNA binding in a subset of other SF2 enzymes, however, the roles of the ARL in PriA had not been investigated. Here, we show that changes within the ARL sequence uncouple PriA ATPase activity from DNA binding. In vitro protein-DNA crosslinking experiments define a residue- and nucleotide-specific interaction map for PriA, showing that the ARL binds replication fork junctions whereas other sites bind the leading or lagging strands. We propose that DNA binding to the ARL allosterically triggers ATP hydrolysis in PriA. Additional SF2 helicases with similarly positioned loops may also couple DNA binding to ATP hydrolysis using related mechanisms.

Function of a strand-separation pin element in the PriA DNA replication restart helicase

“…Arrows highlight the significant shifted bands due to covalent PriA-DNA cross-links. See previous study for expected PriA-DNA cross-link shifts on each strand (20). Representative gel of a single replicate shown out of three.…”

“…E. coli PriA variants were created with the non-natural amino acid Bpa incorporated at 1 of 4 residues at the tip of the ␤-hairpin (PriA Q456 Bpa , A457 Bpa , Q458 Bpa , and H459 Bpa ). Each purified variant was incubated with a synthetic DNA replication fork, then exposed to UV light and assayed for the presence of covalent protein-DNA cross-links by a denaturing EMSA, as has been done previously to map other DNA-binding interfaces in PriA (14,20). A previously published PriA E492 Bpa variant, which also cross-links with the template lagging strand (20) and is positioned near the CRR ␤-hairpin, was analyzed for comparison.…”

“…Each purified variant was incubated with a synthetic DNA replication fork, then exposed to UV light and assayed for the presence of covalent protein-DNA cross-links by a denaturing EMSA, as has been done previously to map other DNA-binding interfaces in PriA (14,20). A previously published PriA E492 Bpa variant, which also cross-links with the template lagging strand (20) and is positioned near the CRR ␤-hairpin, was analyzed for comparison. Four DNA fork types were analyzed with each variant, where each of the four oligonucleotides of the DNA fork were individually radiolabeled.…”

“…WT PriA is the negative control and all lanes were compared back to these due to the presence of partial reannealing. PriA E492 Bpa is a positive control (20). Arrows highlight the significant shifted bands due to covalent PriA-DNA cross-links.…”

“…Despite our advanced understanding of the structure of PriA, the mechanisms by which PriA unwinds DNA remain ill-defined. 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, however, how this activity is linked to strand separation.…”