Ferroplasma acidarmanus RPA2 facilitates efficient unwinding of forked DNA substrates by monomers of FacXPD helicase

Abstract

The strand-separation activity that is important for many cellular DNA processing machineries is provided by DNA helicases. In order to understand the physiological properties of a helicase acting in the context of its macromolecular machinery, it is imperative to identify the proteins that interact with the enzyme and to analyze how these proteins affect its helicase activities. The archaeal Rad3 helicase XPD (xeroderma pigmentosum group D protein) from Ferroplasma acidarmanus (FacXPD) is a superfamily II 5'-->3' DNA helicase. Similar to its mammalian homolog working as an integral part of the transcription factor IIH complex, FacXPD may play an important role in nucleotide excision repair (NER) and transcription initiation. Interaction between FacXPD and other archaeal NER proteins likely modulates their respective activities. Replication protein A (RPA), a single-stranded DNA (ssDNA)-binding protein, is one of the NER proteins that functionally interact with the human transcription factor IIH complex. There are two RPA proteins in F. acidarmanus: FacRPA1, a homodimer of two monomers consisting of two oligonucleotide/oligosaccharide binding folds, and FacRPA2, a monomer containing a single oligonucleotide/oligosaccharide binding fold. In this study, we analyzed the effect of these ssDNA-binding proteins on FacXPD helicase activity. We found that FacRPA2 stimulates DNA unwinding by FacXPD helicase through a novel mechanism by providing a helix-destabilizing function. In contrast, FacRPA1 fails to stimulate helicase activity to the same extent as FacRPA2 and competes with FacXPD for binding to the ssDNA-double-stranded DNA junction. We conclude that the FacRPA2-coated fork is a preferred and likely physiological substrate that a monomer of FacXPD can unwind with a processivity sufficient for expansion of the NER or transcription bubble. We also suggest that duplex melting by a cognate ssDNA-binding protein coordinated with translocation by a helicase may represent a common strategy for duplex unwinding by the Rad3 family of helicases.