Abstract
Helicases catalyse DNA and RNA strand separation. Proteins bound to the nucleic acid must also be displaced in order to unwind DNA. This is exemplified by accessory helicases that clear protein barriers from DNA ahead of advancing replication forks. How helicases catalyse DNA unwinding is increasingly well understood but how protein displacement is achieved is unclear. Escherichia coli Rep accessory replicative helicase lacking one of its four subdomains, 2B, has been shown to be hyperactivated for DNA unwinding in vitro but we show here that RepΔ2B is, in contrast, deficient in displacing proteins from DNA. This defect correlates with an inability to promote replication of protein-bound DNA in vitro and lack of accessory helicase function in vivo. Defective protein displacement is manifested on double-stranded and single-stranded DNA. Thus binding and distortion of duplex DNA by the 2B subdomain ahead of the helicase is not the missing function responsible for this deficiency. These data demonstrate that protein displacement from DNA is not simply achieved by helicase translocation alone. They also imply that helicases may have evolved different specific features to optimise DNA unwinding and protein displacement, both of which are now recognised as key functions in all aspects of nucleic acid metabolism.
Highlights
Helicases perform critical functions in all aspects of nucleic acid metabolism by unwinding and remodelling doublestranded DNA and RNA
In spite of our increasing understanding of how ATP hydrolysis is coupled to the disruption of base pairing, we know very little about how protein displacement from nucleic acids is catalysed
We have investigated the relationship between helicasecatalysed DNA unwinding and protein displacement
Summary
Helicases perform critical functions in all aspects of nucleic acid metabolism by unwinding and remodelling doublestranded DNA (dsDNA) and RNA. Rep is the accessory helicase in Escherichia coli, translocating 3 5 along ssDNA and likely operating on the leading strand template at the replication fork to aid protein displacement ahead of the fork [12,13] This activity is facilitated by a physical and functional interaction between the Rep Cterminus and the 5 -3 replicative helicase DnaB [12,15,16,17]. A PcrA monomer can unwind with high processivity in combination with its partner protein the plasmid initiator RepD [36] which correlates with preferential formation of the closed state of PcrA when bound by RepD [24] It is far less clear how the 2B subdomain affects helicase function in vivo. Given the ubiquity of proteins bound to DNA inside cells, such adaptations are likely to have evolved in other helicases to ensure that protein-bound DNA is unwound effectively when and where required
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