Inching over hurdles: how DNA helicases move on crowded lattices

Abstract

Many of the genome maintenance transactions require continuous progression of molecular motors along single or double stranded DNA (dsDNA) molecule. DNA, however, is rarely found in the cell in its bare form. Structural proteins organize dsDNA and control its accessibility to molecular machines of DNA replication, repair, recombination and transcription. Single-stranded DNA (ssDNA) is sequestered by ssDNA binding proteins, which protect it from degradation, modification and undesired transactions. Appreciation of how molecular machines compete with these stationary blocks and with each other for the access to DNA is important for our understanding of the mechanisms underlying genome maintenance. This understanding in turn establishes the molecular basis of various human diseases resulting from defects in molecular motors and their ability to navigate in crowded intracellular environments. By building upon our recent finding that it is possible for a helicase translocating on ssDNA to bypass a stationary bound protein without displacing it, we discuss potential outcomes of collisions between DNA helicases and ssDNA binding proteins. We then propose that the selective ability of some helicases to bypass or displace a specific ssDNA binding protein may be important for activation of these enzymes for particular DNA maintenance tasks.

Figure 1. Nucleic acid motors and their cellular lattices

(a) Nucleosomes organize the genomic DNA and present a barrier to various cellular DNA transactions. (b) Chromatin remodeling machines regulate access to genetic information by dislodging or repositioning nucleosomes. (c) Nucleosomes may also affect transcription by impeding progression of RNA polymerase. (d) Recombination specific chromatin remodeler, Rad54 is stimulated by interaction with nucleoprotein filament formed by Rad51 recombinase on ssDNA^, . (e) Rad54 also controls postsynaptic steps in homologous recombination by acting on the products of Rad51-catalyzed strand invasion. Several bona fide DNA helicases also act at this step. (f) Srs2 helicase controls recombination by facilitating disassembly of Rad51 nucleoprotein filaments. (g) SMARCAL1 (also known as HARP) is an annealing helicase whose function involves re-formation of the DNA duplex by re-zipping ssDNA coated with ssDNA binding protein, RPA.

Figure 2. Obstacle bypass by XPD helicase

(a) Structure and domain organization of XPD helicase (PDB: 3crv). HD1 and HD2 and helicase domains 1 and 2 respectively. These two domains form SF2 helicase motor core. Arch and FeS are the two family-specific auxiliary domains. (b) One possible configuration of ssDNA within translocating XPD helicase. After the primary binding site (indicated by yellow arrows) ssDNA passes through the hole between Arch and FeS domains into the secondary DNA binding site in the FeS domain. Green arrow illustrates energy transfer from Cy3 fluorophore to the FeS cluster resulting in distance-dependent Cy3 quenching. (c) Alternative configuration of the ssDNA-XPD complex, which does not involve ssDNA passage through the hole between two auxiliary domains. (d) Single-molecule XPD translocation assay exploits FeS-dependent fluorescence quenching (left panel). Representative fluorescence trajectory (middle panel) shows that XPD translocation along ssDNA decorated with Cy3 dye at the 3′-end results in gradual quenching of Cy3 intensity followed by its full recovery when XPD dissociates from the substrate. Distribution of individual translocation rates binned in 5% fluorescence change per second intervals (right panel). (e) Single-molecule experiment carried out in the presence of RPA2 protein. (f) Simultaneous visualization of XPD translocation (monitored by following FeS-dependent quenching of Cy3 dye) and RPA2 binding (monitored by following FRET between Cy3 dye incorporated at the 3′-end of ssDNA and Cy5 dye located at the N-terminus of RPA2 protein). Fluorescence of the Cy3 and Cy5 dyes is shown in green and red, respectively. A synergistic quenching followed by the recovery of Cy3 and Cy5 fluorescence suggested that XPD helicase can bypass bound RPA without dissociating it from the lattice. (g.) Translocation of XPD was observed through quenching of directly excited Cy5-RPA2. Gradual decrease and increase in Cy5 intensity reflected XPD approaching and moving away from Cy5 labeled RPA2. We interpreted this quenching pattern as translocation of XPD helicase over bound RPA2. Data shown in this figure are adapted from Honda, et al.

Figure 3. Scenarios for co-existence of a helicase and ssDNA binding protein on the DNA substrate

(a) Targeting of a helicase to an ssDNA-dsDNA junction. (b) A helicase can step-over the region of ssDNA wrapped around an SSB. (c) Physical interaction between helicase and SSB may facilitate bypass and prevent dissociation of SSB. (d) Helicase can bypass bound SSB without displacing it from ssDNA if the two proteins interact with different features on DNA (for example, with phosphodiester backbone and nitrogen bases).