Molecular traffic jams on DNA

Abstract

All aspects of DNA metabolism-including transcription, replication, and repair-involve motor enzymes that move along genomic DNA. These processes must all take place on chromosomes that are occupied by a large number of other proteins. However, very little is known regarding how nucleic acid motor proteins move along the crowded DNA substrates that are likely to exist in physiological settings. This review summarizes recent progress in understanding how DNA-binding motor proteins respond to the presence of other proteins that lie in their paths. We highlight recent single-molecule biophysical experiments aimed at addressing this question, with an emphasis placed on analyzing the single-molecule, ensemble biochemical, and in vivo data from a mechanistic perspective.

Figure 1

DNA replication, transcription, and repair must occur simultaneously on a crowded nucleic acid substrate. Conflicts between these molecular machines must be resolved rapidly to maintain cell viability and to avoid genomic instability.

Figure 2

(a) An illustration of the single-molecule DNA curtain assay used to observe RecBCD-roadblock collisions. Individual DNA molecules are tethered to a fluid lipid bilayer via a biotin-streptavidin interaction and organized at nanofabricated chrome barriers. RecBCD is loaded at the free DNA ends and fluorescent RNA polymerase (RNAP) is deposited on the native promoters along the DNA. The DNA is visualized by YOYO1, an intercalating fluorescent dye that does not interfere with RecBCD activity. The reaction is initiated by supplementing the flow buffer with ATP. As the DNA is degraded by RecBCD helicase/nuclease activity, the time-dependent decrease in DNA length serves as readout of RecBCD translocation. (b) Kymographs of RecBCD pushing and evicting RNAP holoenzyme and elongation complexes (ECs) from DNA. In all kymographs, the tethered end of the DNA is at the top, the free end is at the bottom, and buffer flow is from top to bottom. RecBCD is able to efficiently push and eventually displace RNAP holoenzyme (top panel ), transcribing ECs (bottom panel ). (c) A scatter plot of pre- and postcollision RecBCD velocities. The red line is a fit to the data with a slope of 1.0. RecBCD does not change velocity upon collision with RNAP. (d ) A transition-state ejection model for roadblock displacement by RecBCD. Most roadblocks are displaced from RecBCD as they are pushed from one nonspecific DNA site to the next. Translocating RecBCD initially displaces the protein roadblock from a high-affinity specific site (S). The roadblock must pass through a much more weakly bound intermediate state (T) as it is pushed from one site to the next, followed by re-equilibration as a DNA-bound nonspecific complex (N). Subsequent steps by RecBCD continue to push the protein roadblocks from one nonspecific site to the next, until the proteins are eventually evicted from the DNA.

Figure 3

(a) A smFRET-based assay for observing the RecA-clearing activity of PcrA helicase. A DNA oligonucleotide with a Cy3-Cy5 FRET dye pair is affixed to the surface of a passivated microscope slide. Entropically driven collapse of the ssDNA brings the two dyes within efficient FRET distance. Addition of RecA leads to a rigid nucleoprotein filament, which separates the two dyes and reduces the observed FRET signal. In the presence of ATP, PcrA helicase clears RecA and reels the free DNA ends closer together. (b) smFRET data. (Top) Naked ssDNA brings both dyes relatively close, leading to a FRET signal of ~0.5. (Middle) Addition of RecA elongates the ssDNA tail and reduces the smFRET to ~0.3. (Bottom) In the presence of ATP, PcrA clears RecA from ssDNA as it reels the two DNA ends close to each other.

Figure 4

A single-molecule dye-quenching assay for XPD translocation along replication protein A (RPA)-coated DNA. (a) A DNA oligonucleotide is immobilized on the surface of a passivated flowcell and decorated with Cy5-labeled RPA. The FeS cluster of XPD helicase quenches Cy5 fluorescence in a distance-dependent manner. (b) When ATP is added, XPD translocation toward RPA initially quenches the Cy5 fluorescence (arrow 1), and eventually XPD bypasses the RPA (arrow 2), which is revealed as an eventual recovery of the Cy5 intensity. (c) Examples of intensity trajectories showing XPD approaching (arrow 1) and moving past (arrow 2) RPA.

Figure 5

(a) Accessory helicases interact with the replisome to remove protein roadblocks. In Escherichia coli, the C terminus of the accessory, nonreplicative Rep helicase physically interacts with DnaB. Accessory helicases can aid replication past protein roadblocks such as LacI repeats, hydrolytically inactive EcoRI, and head-on RNAP collisions. (b) Upon encountering a backtracked RNA polymerase (RNAP) in a codirectional orientation, the replisome can reinitiate from the 3′ end of the mRNA R-loop. A subsequent round of replication converts the resulting nick into a double-strand break. Abbreviation: EC, elongation complex.

Figure 6

An optical trap assay to investigate RNA polymerase II (RNAP II) transcription past a nucleosome. (a) A schematic of the DNA construct used in this study. One end of the DNA was affixed to a polystyrene bead via a DIG–anti-DIG interaction. Biotinylated pol II was assembled onto a promoter (blue) and affixed to a second streptavidin-coated bead. A nucleosome was reconstituted on the strong nucleosome-positioning Widom 601 DNA sequence (brown). (b) An illustration of the experimental setup. RNAP transcription was monitored as a change in the DNA length between the two optical traps. (c) Individual pol II transcription traces as a function of NaCl concentration. At low salts, pol II stalls at the nucleosome barrier. As NaCl concentration is increased, pol II transiently pauses and eventually transcribes past the nucleosome. (d ) A histogram of nucleosome-dependent arrest probability. The polymerase pauses as it encounters the nucleosome but can transcribe past transiently disrupted histone-DNA interactions at higher salt concentrations. (c,d ) Transcription traces on naked DNA are shown in black; transcription traces on DNA with nucleosomes are shown in colors. The shaded regions of the graphs represent the location of the nucleosome positioning sequence. Abbreviations: EC, elongation complex; DIG, digoxigenin.

Figure 7

Two RNA polymerases (RNAPs) collaborate to overcome a nucleosome barrier. An optical tweezers–based assay was used to map the position of a T7 RNAP active site after it encountered a nucleosome. (a) Individual RNAPs stall and backtrack at a nucleosome barrier. The DNA contained a single T7 RNAP promoter (blue) and a strongly positioned nucleosome (brown). Upon encountering the nucleosome, RNAP stalled and backtracked 75 bp away from the nucleosome dyad. Almost no read-through transcription was seen after a 10-s incubation. (b) A trailing RNAP was introduced on a second promoter. The leading RNAP no longer backtracked and paused 60 bp away from the nucleosome dyad. A large fraction of RNAP molecules was able to transcribe through the nucleosome barrier.