Fork sensing and strand switching control antagonistic activities of RecQ helicases

Abstract

RecQ helicases have essential roles in maintaining genome stability during replication and in controlling double-strand break repair by homologous recombination. Little is known about how the different RecQ helicases found in higher eukaryotes achieve their specialized and partially opposing functions. Here, we investigate the DNA unwinding of RecQ helicases from Arabidopsis thaliana, AtRECQ2 and AtRECQ3 at the single-molecule level using magnetic tweezers. Although AtRECQ2 predominantly unwinds forked DNA substrates in a highly repetitive fashion, AtRECQ3 prefers to rewind, that is, to close preopened DNA forks. For both enzymes, this process is controlled by frequent strand switches and active sensing of the unwinding fork. The relative extent of the strand switches towards unwinding or towards rewinding determines the predominant direction of the enzyme. Our results provide a simple explanation for how different biological activities can be achieved by rather similar members of the RecQ family.

Figure 1. DNA hairpin unwinding by AtRECQ2.

(a) Force induced hairpin unzipping. Increasing the force applied to the hairpin construct (see Supplementary Fig. S1b) leads initially to slight stretching of the dsDNA handles of the construct. At a critical force (F_unzip=11.6 pN) the 40 bp long hairpin unzips in a single abrupt step followed by further slight extension increases. (b) Typical unwinding event of AtRECQ2 on a 40 bp hairpin in the presence of ATP at a constant force of 10 pN. After slow hairpin unwinding an abrupt rezipping of the hairpin occurs followed by a resetting, that is, the reinititation of a new unwinding event (see sketches below). (c) Global unwinding pattern of AtRECQ2 on the 40 bp DNA hairpin under the same conditions as in b. (upper) Individual saw tooth-like events of similar size occur in a highly repetitive fashion. (lower). Periods of highly repetitive unwinding events are interspersed with periods of no activity.

Figure 2. Behaviour of AtRECQ2 on single-stranded DNA.

(a) Measuring the distance AtRECQ2 moved on ssDNA by repetitive cycles of hairpin unzipping. A 488 bp long DNA hairpin (F_unzip=15.8 pN, upper graph red solid line) is mechanically un- and rezipped by switching the force between F=6.6 pN (hairpin closed) and F=16.8 pN (hairpin open). At the low force, a slowly increasing DNA extension indicates hairpin unwinding by the helicase. Switching to the high force unzips the hairpin, seen as a sudden large increase of the DNA extension. The hairpin is kept open for the time τ_open, after which the force is lowered and the hairpin rezips until it encounters the helicase. The distance that the enzyme moved on the unzipped ssDNA (h_ssDNA) is then the difference of the hairpin lengths just after and just before hairpin re- and unzipping, respectively. (b) Histograms of h_ssDNA for increasing τ_open (as given in the plots). The red dotted line indicates h_ssDNA=0. The yellow area indicates the region within the s.d. of the distribution around its mean. Only events for which AtRECQ2 did not reach the end of the hairpin were considered. (c) Model for repetitive DNA unwinding by AtRECQ2 of the 40 bp hairpin. After full unwinding of the DNA hairpin the enzyme becomes more loosely bound to ssDNA. After it moves over the hairpin loop, the rehybridizing hairpin pushes the helicase along the formerly displaced strand until the hairpin is closed. Subsequently the helicase switches back to the original tracking strand and initiates a new unwinding event.

Figure 3. Partial rewinding and direction reversals of AtRECQ2.

(a–c) Selected unwinding events recorded on the 40 bp DNA hairpin. Dashed lines indicate the position of the fully closed and the fully open DNA hairpin. The scale bars indicate 1 s. The small roman numbers in the graphs correspond to the following types of resetting event as illustrated with sketches below the graphs: (i) continuous, abrupt hairpin rezipping; (ii) abrupt rezipping and partial slow rewinding; and (iii) direction reversals from rewinding to unwinding. In (a,b) events in which the hairpin is fully unwound, which were recorded at forces of 7 and 11 pN, respectively, are depicted. At the high force the hairpin spontaneously unzips shortly before full unwinding and may stay open for a longer period. In c, incomplete unwinding events taken at 7 pN are shown. (d) Bulk unwinding experiments. Used DNA substrates are sketched above each graph. In all, 0.15 nM DNA was incubated with decreasing concentrations (as illustrated by the wedge symbol) of AtRECQ2 (18, 6 and 2 nM) or AtRECQ3 (25, 8 and 3 nM) and with 19 nM AtRECQ2-K117M or 18 nM AtRECQ3-K64M (indicated with c) in the presence of ATP. Reaction products were analysed by native gel electrophoreses and the amount of DNA unwinding determined. The error bars represent the s.d. of four individual measurements. nts, nucleotides.

Figure 4. Clamping of the unwinding fork by AtRECQ2.

(a) Repetitive mechanical unzipping of a 488 bp long hairpin in absence of AtRECQ2 by switching the force between 10.4 and 18 pN. The red solid line in the upper graph indicates the unzipping force. (b) Repetitive force switching between 10.4 and 18 pN for the hairpin in a during unwinding by AtRECQ2. White areas indicate periods of low force. Coloured areas represent periods at high force during which the hairpin fully unzips (red) or remains fully closed (green). (c) Same experiment as in b for force switches between 10.4 and 16 pN. (d) DNA unwinding of the 488 bp long hairpin by AtRECQ2 at 10 pN force. The positions of the fully closed and the fully opened DNA hairpin are indicated with black solid lines. Abrupt rezipping events starting before the full hairpin is unwound and ending before it is fully closed are indicated with light grey boxes. (e) Histogram of the distances of abrupt rezipping events for which the hairpin does not fully close (shown in grey, see D for details). For comparison, a histogram of the negative moved distances on ssDNA (h_ssDNA, from the experiments shown in Fig. 2a for hairpin opening times <3 s) is shown in blue. nt, nucleotide.

Figure 5. DNA unwinding and rewinding by AtRECQ3.

(a) Unwinding pattern of AtRECQ3 on the 488 bp DNA hairpin in the presence of ATP at constant force of 10 pN. (upper) Slow DNA unwinding is always followed by slow DNA rewinding. (middle) Individual unwinding–rewinding events of similar extent occur in a highly repetitive fashion. The AtRECQ3 catalysed DNA unwinding is much shorter than the full-length of the hairpin. (lower) Periods of highly repetitive unwinding events are interspersed with periods of no activity. (b) Unzipping and rezipping the 488 bp DNA hairpin during activity of AtRECQ3. Corresponding force switches are shown in the top graph. The red line indicates F_unzip. In contrast to the very short unwinding distances found for AtRECQ3, the DNA hairpin is processively rewound over hundreds of bp (events indicated by arrows). (c) Enlarged view into an individual rewinding event reveals permanent direction reversals from rewinding to unwinding and back to rewinding.

Figure 6. Unified model for repetitive DNA unwinding by AtRECQ2 and AtRECQ3.

The helicase is sketched in orange with its tip representing the RecQ-C-terminal domain, where the bp opening is catalysed. The red and green rectangles represent the two RecA-like domains. Uncoloured RecA-like domains symbolize low affinity to the ssDNA during sliding. During unwinding, the DNA junction is clamped symbolized by the enzyme-captured displaced strand. After binding to a 3′ overhang (A), the helicases start to processively unwind the duplex DNA by tracking along the 3′–5′ direction (B). The unwinding reaction stops either due to a spontaneous strand switch from the tracking to the displaced strand (C) or due to the full unwinding of the DNA hairpin (C*). The helicases may then continue to slowly translocate along the formerly displaced strand (D). This leads to slow rewinding of the hairpin, as it reanneals behind the helicase. Alternatively, AtRECQ2 may also rapidly slip back along the displaced strand while being pushed by the reannealing hairpin (D*). As shown above, AtRECQ2 is not only translocating on ssDNA but can in contrast to AtRECQ3 adopt a weaker bound diffusive state (Fig. 2), which supports the slippage. After rewinding or, respectively, resetting, the enzyme switches back from the formerly displaced (the actual tracking strand) to the original tracking strand (E) and reinitiates a new unwinding cycle.