Helicases and nucleic acid translocases are motor proteins that have essential roles in nearly all aspects of nucleic acid metabolism, ranging from DNA replication to chromatin remodelling. Fuelled by the binding and hydrolysis of nucleoside triphosphates, helicases move along nucleic acid filaments and separate double-stranded DNA into their complementary single strands. Recent evidence indicates that the ability to simply translocate along single-stranded DNA is, in many cases, insufficient for helicase activity. For some of these enzymes, self assembly and/or interactions with accessory proteins seem to regulate their translocase and helicase activities.

Escherichia coli RecBCD is a DNA helicase with two ATPase motors (RecB, a 3′ to 5′ translocase, and RecD, a 5′ to 3′ translocase) that functions in repair of double-stranded DNA breaks. The RecBC heterodimer, with only the RecB motor, remains a processive helicase. Here we examined RecBC translocation along single stranded (ss) DNA. Surprisingly, we find that RecBC displays two translocase activities: the primary translocase moves 3′ to 5′, while the secondary translocase moves RecBC along the opposite strand of a forked DNA at a similar rate. The secondary translocase is insensitive to the ssDNA backbone polarity, and we propose that its function may be to fuel RecBCD translocation along double stranded DNA ahead of the unwinding fork, and to ensure that the unwound single strands move through RecBCD at the same rate after interaction with a Chi sequence.

Guanine rich nucleic acid sequences can form G-quadruplex (G4) structures that interfere with DNA replication, repair and RNA transcription. The human FANCJ helicase contributes to maintaining genomic integrity by promoting DNA replication through G4-forming DNA regions. Here, we combined single-molecule and ensemble biochemical analysis to show that FANCJ possesses a G4-specific recognition site. Through this interaction, FANCJ targets G4-containing DNA where its helicase and G4-binding activities enable repeated rounds of stepwise G4-unfolding and refolding. In contrast to other G4-remodeling enzymes, FANCJ partially stabilizes the G-quadruplex. This would preserve the substrate for the REV1 translesion DNA synthesis polymerase to incorporate cytosine across from a replication-stalling G-quadruplex. The residues responsible for G-quadruplex recognition also participate in interaction with MLH1 mismatch-repair protein, suggesting that the FANCJ activity supporting replication and its participation in DNA interstrand crosslink repair and/or heteroduplex rejection are mutually exclusive. Our findings not only describe the mechanism by which FANCJ recognizes G-quadruplexes and mediates their stepwise unfolding, but also explain how FANCJ chooses between supporting DNA repair versus promoting DNA replication through G-rich sequences.

Regulation of translocation polarity by helicase domain 1 in SF2B helicasesBiochemical and reverse footprinting studies of the nucleotide excision repair protein XPD show that opposing translocation polarity in superfamily II A and B helicases is an intrinsic property of their respective motor domains, rather than related to different relative DNA binding orientations.

Background: RecBCD helicase is involved in repair of double-stranded DNA breaks. Results: The 5Ј to 3Ј ssDNA translocation rate of RecBCD is faster than the 3Ј to 5Ј rate in the absence of a CHI site, and the rates are coupled asymmetrically. Conclusion: RecBC controls 3Ј to 5Ј and 5Ј to 3Ј translocation, but RecD controls only 5Ј to 3Ј translocation. Significance: Asymmetric regulation may explain how RecBCD is regulated after CHI recognition.