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.

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.

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.

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.

E. coli RecBCD is a bipolar DNA helicase possessing two motor subunits (RecB, a 3' to 5' translocase and RecD, a 5' to 3' translocase), that is involved in the major pathway of recombinational repair. Previous studies indicated that the minimal kinetic mechanism needed to describe the ATP-dependent unwinding of blunt-ended DNA by RecBCD in vitro is a sequential n-step mechanism with two to three additional kinetic steps prior to initiating DNA unwinding. Since RecBCD can “melt-out” ~ six bp upon binding to the end of a blunt-ended DNA duplex in a Mg2+-dependent but ATP-independent reaction, we have investigated the effects of non-complementary single-stranded (ss) DNA tails (3'-(dT)6 and 5'-(dT)6 or 5'-(dT)10) on the mechanism of RecBCD and RecBC unwinding of duplex DNA using rapid kinetic methods. As with blunt-ended DNA, RecBCD unwinding of DNA possessing 3'-(dT)6 and 5'-(dT)6 non-complementary ssDNA tails is well described by a sequential n-step mechanism with the same unwinding rate (mkU = 774 ± 16 bp s−1) and kinetic step-size (m = 3.3 ± 1.3 bp), yet two to three additional kinetic steps are still required prior to initiation of DNA unwinding (kC = 45 ± 2 s−1). However, when the non-complementary 5'-ssDNA tail is extended to ten nucleotides (5'-(dT)10 and 3'-(dT)6), the DNA end structure for which RecBCD displays optimal binding affinity, the additional kinetic steps are no longer needed, although a slightly slower unwinding rate (mkU = 538 ± 24 bp s−1) is observed with a similar kinetic step-size (m = 3.9 ± 0.5 bp). The RecBC DNA helicase (without the RecD subunit) does not initiate unwinding efficiently from a blunt DNA end. However, RecBC does initiate well from a DNA end possessing non-complementary twin 5'- dT6 and 3'-dT6 tails, and unwinding can be described by a simple uniform n-step sequential scheme, without the need of the additional kC initiation steps, with a similar kinetic step size (m = 4.4 ± 1.7 bp), and unwinding rate (mkobs = 396 ± 15 bp s−1). These results suggest that the additional kinetic steps with rate constant kC required for RecBCD to initiate unwinding of blunt-ended and twin (dT6)–tailed DNA reflect processes needed to engage the RecD motor with the 5' ssDNA.

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DNA helicases participate in virtually all aspects of cellular DNA metabolism by using ATP-fueled directional translocation along the DNA molecule to unwind DNA duplexes, dismantle nucleoprotein complexes, and remove non-canonical DNA structures. Post-translational modifications and helicase interacting partners are often viewed as determining factors in controlling the switch between bona fide helicase activity and other functions of the enzyme that do not involve duplex separation. The bottleneck in developing a mechanistic understanding of human helicases and their control by post-translational modifications is obtaining sufficient quantities of the modified helicase for traditional structure-functional analyses and biochemical reconstitutions. This limitation can be overcome by single-molecule analysis, where several hundred surface-tethered molecules are sufficient to obtain a complete kinetic and thermodynamic description of the helicase-mediated substrate binding and rearrangement. Synthetic oligonucleotides site-specifically labeled with Cy3 and Cy5 fluorophores can be used to create a variety of DNA substrates that can be used to characterize DNA binding, as well as helicase translocation and duplex unwinding activities. This chapter describes “single-molecule sorting”, a robust experimental approach to simultaneously quantify, and distinguish the activities of helicases carrying their native post-translational modifications. Using this technique, a DNA helicase of interest can be produced and biotinylated in human cells to enable surface-tethering for the single-molecule studies by total internal reflection fluorescence microscopy. The pool of helicases extracted from the cells is expected to contain a mixture of post-translationally modified and unmodified enzymes, and the contributions from either population can be monitored separately, but in the same experiment providing a direct route to evaluating the effect of a given modification.