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. Author manuscript; available in PMC: 2014 Sep 12.
Published in final edited form as: Mol Cell. 2013 Aug 22;51(5):691–701. doi: 10.1016/j.molcel.2013.07.016

PICH: a DNA Translocase Specially Adapted for Processing Anaphase Bridge DNA

Andreas Biebricher 1,#, Seiki Hirano 2,#, Jacqueline H Enzlin 3, Nicola Wiechens 4, Werner W Streicher 5, Diana Huttner 3,5, Lily H-C Wang 6,&, Erich A Nigg 6, Tom Owen-Hughes 4, Ying Liu 3, Erwin Peterman 1,#, Gijs JL Wuite 1,#, Ian D Hickson 2,3,#
PMCID: PMC4161920  EMSID: EMS60325  PMID: 23973328

SUMMARY

The PICH protein localises to ultra-fine anaphase DNA bridges (UFBs) in mitosis alongside a complex of DNA repair proteins, including BLM. However, very little is known about the function of PICH or how it is recruited selectively to UFBs. Using a combination of microfluidics, fluorescence microscopy and optical tweezers, we have defined the properties of PICH in an in vitro model of an anaphase bridge. We show that PICH binds with a remarkably high affinity to dsDNA, resulting in ATP-dependent protein translocation and extension of the DNA. Most strikingly, the affinity of PICH for binding dsDNA increases with tension-induced DNA stretching, which mimics the effect of the mitotic spindle on a UFB. PICH binding also appears to diminish force-induced DNA melting. We propose a new model in which PICH recognizes, binds to and stabilizes DNA under tension during anaphase, thereby facilitating the resolution of entangled sister chromatids prior to cytokinesis.

INTRODUCTION

The maintenance of chromosome integrity depends critically upon the accurate replication of the genome in S-phase and the faithful segregation of sister chromatids in M-phase. A failure to efficiently execute sister chromatid disjunction can lead to the formation of anaphase DNA bridges and micronuclei (Lengauer et al., 1998). Mitotic chromosome nondisjunction can also generate aneuploidy, a near universal characteristic of solid tumours in humans (Gisselsson, 2003). Although it was thought for decades that anaphase DNA bridging was a very rare event during an unperturbed cell cycle, recent evidence has overturned this view (Baumann et al., 2007; Chan et al., 2007). It is now established that most mitoses are associated with the formation of so-called ultra-fine anaphase DNA bridges (UFBs), which had escaped detection previously because they cannot be stained with commonly-used DNA dyes (e.g. DAPI) and do not appear to contain histones (Baumann et al., 2007; Chan and Hickson, 2011; Chan et al., 2007). UFBs are readily detectable in early anaphase, but their number declines sharply as anaphase progresses, suggestive of active resolution in anaphase itself (Baumann et al., 2007; Chan et al., 2007).

Most UFBs originate from centromeric DNA (Baumann et al., 2007; Chan et al., 2007). This perhaps reflects the intertwining (catenation) of the DNA between sister-centromeres that contributes to the maintenance of centromeric cohesion until the metaphase to anaphase transition is triggered by the destruction of the cohesin complex (Kaulich et al., 2012; Wang et al., 2010; Wang et al., 2008). Although this putative physiological role for UFBs is plausible, it is clear that some UFBs arise from common fragile site loci, and these UFBs very likely arise due to pathological DNA transactions (Chan and Hickson, 2011; Chan et al., 2009; Naim and Rosselli, 2009). Fragile sites are regions of the genome prone to breakage in metaphase, and are thought to represent loci where the completion of DNA replication is delayed or problematic (Durkin and Glover, 2007). Both classes of UFB (centromeric and fragile site-associated) can be visualized via immuno-fluorescence using antibodies to either the PICH protein or any of the so-called BTRR protein complex comprising BLM, Topoisomerase IIIα, RMI1 and RMI2 (Baumann et al., 2007; Chan and Hickson, 2011; Chan et al., 2007; Wang et al., 2010; Ying and Hickson, 2011). BLM is the protein defective in Bloom’s syndrome (BS), a rare condition associated with dramatic cancer predisposition (German, 1993). At the cellular level, BS is characterised by genomic instability, including an elevated frequency of sister chromatid exchanges, micronuclei and anaphase bridging (Chu and Hickson, 2009).

PICH is an SNF2 family member that was identified initially as a binding partner of the mitotic regulatory kinase, PLK1 (Baumann et al., 2007). There have been several phenotypes reported for mammalian cells depleted of PICH. These include effects on mitotic checkpoints and metaphase chromosome architecture, but many of these phenotypes were not seen in all studies, probably due to off-target effects of the siRNAs (Baumann et al., 2007; Kurasawa and Yu-Lee, 2010; Leng et al., 2008). Recent data, derived from the use of more specific siRNAs or the microinjection of anti-PICH antibodies, indicate that impairment of PICH function leads to an elevation in the frequency of bulky (DAPI-positive) anaphase bridges (Hubner et al., 2010). It has also been proposed previously that the role of PICH is to remodel nucleosomes on DNA, a characteristic activity of some SNF2 family members (Ke et al., 2011). This would be a plausible role, given that UFBs seem to lack histones (Baumann et al., 2007; Chan et al., 2007). However, PICH also binds similarly to chromatinized anaphase bridges (Baumann et al., 2007; Chan et al., 2007), suggesting that PICH is unlikely to act as a simple histone ‘evictor’. What is clear is that PICH generally coats UFBs along their entire length, irrespective of whether the cell is in early or late anaphase, and its binding is therefore not confined to any region of the UFB such as the location of the presumed catenated DNA. The BTRR complex also coats UFBs along their length, but this association is dependent upon PICH (Chan and Hickson, 2011; Chan et al., 2007; Ke et al., 2011; Ying and Hickson, 2011).

In order to understand the dynamics of UFB processing by PICH, we have monitored PICH interactions with DNA in real time. Using an instrument that combines single-molecule fluorescence microscopy with optical trapping, we created a laboratory mimic of a cellular anaphase that contains a PICH-bound UFB. Using this set-up, we have tested whether PICH has an inherent ability to recognize features of DNA that might exist uniquely in a UFB. It was of particular interest to test the hypothesis that PICH has an inherent bias in its binding to DNA molecules that are under tension, as might be expected for UFBs tethered at each end by sister chromatids being progressively pulled in opposite directions as anaphase proceeds.

RESULTS

Purification of different forms of PICH protein

To analyze the biochemical and biophysical properties of the PICH protein, we expressed four versions of recombinant human PICH in insect cells and purified the proteins to homogeneity (Figure S1A). Two of these proteins were non-fluorescent versions for use in ensemble studies, and were designated native PICH and PICH-K128A (with a substitution of the essential lysine required for ATP binding). The other two, designated PICH-eGFP and eGFP-PICH, were fluorescent variants for use in single molecule studies.

PICH is a DNA-dependent ATPase and an ATP-dependent translocase

First, we analyzed the native PICH and PICH-K128A proteins for their ability to hydrolyze ATP. PICH displayed a robust, DNA-dependent ATPase activity that was, as expected, absent from the PICH-K128A protein (Figure S1B). This result indicates that ATPase activity is an intrinsic property of PICH, and not due to a protein contaminant in the PICH preparation. SNF2 family proteins generally utilize the energy derived from ATP hydrolysis to translocate on dsDNA, but are incapable of translocating along ssDNA and cannot, therefore, act as helicases to separate the complementary strands of a DNA duplex (Enemark and Joshua-Tor, 2008; Pazin and Kadonaga, 1997). As expected, and as shown in an earlier report (Ke et al., 2011), PICH lacked DNA strand separating activity (unlike BLM, a positive control; Figure S1C). To analyze PICH’s ability to act as a dsDNA translocase, we employed the conventional DNA triplex assay in which the displacement of a radioactively labelled triplex-forming oligonucleotide from a DNA duplex is monitored. Native PICH, but not PICH-K128A, was able to catalyze triplex displacement in a concentration- and time-dependent manner (Figure 1A/B). We then engineered various changes into the structure of the triplex-forming oligonucleotide or the duplex DNA (Figure S2A) to assess how this might affect PICH’s translocation function. We found that fully annealed and partially annealed oligonucleotides could be displaced (Figure S2A), as could an oligonucleotide annealed to short duplex (60 bp) (Figure S2B). These data indicate that the loading of PICH to DNA can occur when the free dsDNA region is only 20 bp long. Moreover, PICH does not require a free DNA end on the dsDNA in order to initiate translocation, as revealed by its ability to displace a triplex annealed to a circular plasmid molecule (Figure S2C). Consistent with the above data, electrophoretic mobility shift assays indicated that PICH formed a stable complex with dsDNA, but not ssDNA (Figure S2D). This binding was not compromised by substitution of the Walker A-box lysine residue.

Figure 1. PICH is an ATP-dependent translocase protein.

Figure 1

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(A) Representative time course (as indicated above the lanes) of DNA triplex disruption by native PICH at either 2 nM (top panel) or 8 nM (middle), and of PICH-K128A at 8 nM (bottom). Lane ‘H’ denotes heat-denatured substrate. The positions of the triplex substrate and the free ssDNA oligonucleotide are shown on the right. The red asterisk denotes the radioactive label. The experiment was repeated 3 times with comparable results. (B) Quantification of the data from panel A. See also Figures S1, S2, S3, S4 and S5.

We also demonstrated that PICH could translocate a Holliday junction, a defined intermediate in homologous recombination. This was analyzed because RAD54, a SNF2 family member with significant sequence similarity to PICH (Ceballos and Heyer, 2011; Tan et al., 2003), has been reported to catalyze Holliday junction branch migration (Mazin et al., 2010). Through analysis of the widely used ‘X-12’ mimic of a Holliday junction (Whitby and Lloyd, 1998), we showed that PICH can promote branch migration of the 4-way junction into the characteristic ‘splayed arm’ products (Figure S3A-C). This activity was, as expected, absent from PICH-K128A protein.

PICH does not show a significant ability to remodel nucleosomes

Recent data suggest that PICH might display remodelling activity of histones on anaphase-bridge DNA (Ke et al., 2011), which would be consistent with the elevated frequency of histone-associated anaphase bridges seen in PICH-depleted cells (Kaulich et al., 2012; Ke et al., 2011). Therefore, we analyzed the ability of our PICH preparations to reposition nucleosomes. Typically, such activity can be detected using model templates on which nucleosomes are assembled at either the centre or ends of short DNA fragments. To test the activity of PICH, we used nucleosomes assembled at the end (0w47), or centre (42×47, 54A54) using canonical histones, or octamers incorporating the centromeric histone variant CENP-A (as most UFBs are centromere-derived). Although we analyzed several independent PICH preparations, including native PICH and the PICH-eGFP preparations utilized in the single-molecule studies (see below), we were unable to detect any effect of PICH on nucleosome position or stability on the DNA (Figure S4). Hence, we conclude that PICH is unlikely to function in nucleosome remodelling of regular chromatin, at least not in the absence of other co-factors or DNA tension (see Discussion).

Direct visualization of PICH-DNA interactions

To visualize the interaction of PICH with DNA in real time, we utilized a combination of double-trap optical tweezers, fluorescence microscopy and micro-fluidics. This approach allowed us to visualize eGFP-labeled PICH proteins as they interacted with a phage λ DNA molecule tethered at each end by a trapped polystyrene microsphere, mimicking a UFB under tension (Figure 2A-C). Most of the experimental results presented were obtained using PICH-eGFP; however, we always verified that comparable results were obtained using the eGFP-PICH variant (see Figure S5A/B). We found that PICH displayed a remarkably high affinity for dsDNA, such that extremely low concentrations of PICH (<100 pM) could be utilized for single-molecule studies. Accordingly, we were able to follow the binding and dissociation of PICH from the DNA in real time (Figure 2B and Movie S1). Altogether, the experimental set-up allowed the visualization of single fluorescent proteins with sub-second time resolution and a spatial resolution of 30 nm, while the tension applied to the DNA was being controlled with an accuracy of ±0.1 pN.

Figure 2. Single molecule analysis.

Figure 2

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(A) Outline of single molecule experimental set-up. Experiments were carried out in a four-channel flow cell (top left: cut-out image of cell). In section I (expanded on the right), the laminar flow (indicated by right arrows) prevents the contents in the combined channels from mixing. By moving the flow cell with the aid of a stage, the channels can be rapidly switched to pick up beads (1), capture a single DNA molecule between the beads (2), and check the integrity of the DNA (3). The trapped DNA is subsequently moved into the fourth channel (4) (section II; expanded below), which contains fluorescent PICH in the same buffer. (B) Unprocessed fluorescence image depicting five single PICH proteins bound to λ DNA (invisible) stretched between the beads. See also Movie S1. (C) The fluorescence signal (red boxed area) of a fully PICH-eGFP-coated DNA divided by the average brightness of a single molecule yields the number of bound molecules (~2400) from which the size of the footprint can be derived. (D) The majority of observed PICH-DNA interactions fall into a narrow brightness range that is fitted well by a Gaussian distribution. The small fraction (<5%) of events at significantly higher brightness can be attributed to dimer interactions. (E) The course of the fluorescence signal over time for single binding events under bleaching conditions can be used to distinguish between monomer (bleaching occurs in single steps, traces I-III) and dimer binding (two steps, IV).

Stoichiometry and footprint of the PICH-DNA interaction

eGFP-labeling of proteins provides a straightforward way to determine the stoichiometry of interactions with DNA because it ensures that each bound protein carries a single label. We measured the average fluorescence intensity from multiple PICH-DNA interactions, and found that ~95% of the events displayed a narrow brightness range, which could be fitted to a single Gaussian distribution (Figure 2D). We then conducted experiments under conditions where the eGFP undergoes a destructive photoreaction (bleaching) (Figure 2E). We found that bleaching generally occurred in a single step, a feature typical of single dyes. Only in ~5% of the cases were two bleaching steps observed. We thus conclude that the major fluorescent population likely corresponds to PICH monomers.

The eGFP labeling of PICH also allowed us to estimate the area of the DNA that is covered by the protein (the ‘footprint’). By employing a higher PICH protein concentration (75 nM), we could saturate the DNA binding (Figure 2C). From the calibrated single eGFP fluorescence intensity, we calculated the number of PICH monomers per λ DNA molecule to be 2400 ± 300. From this, we deduced a DNA footprint for PICH of 17 bp ± 3 bp (equivalent to ~6 nm), a value consistent with our finding that loading of PICH to DNA can occur when the free dsDNA region is only 20 bp in length (Figure S2B).

Dynamics of PICH binding to DNA

Next, we determined the DNA binding and dissociation characteristics of PICH by real-time imaging. Using kymographs derived from the image sequences, we determined the protein off-rate by counting the duration of individual protein-binding events (Figure 3A and Movie S1/2). The cumulative distribution of dissociation times could be fitted with a single exponential decay (Figure S6A). Moreover, we found that a decrease in the concentration of monovalent salt from 100 mM to 25 mM NaCl led to an increase of the interaction time of PICH with DNA by a factor of ~20 (18 s to 360 s; see Supplemental Information and Figure S6). We propose, therefore, that the binding of PICH to DNA is predominantly charge-controlled and likely mediated via the phosphate backbone.

Figure 3. Translocation by PICH.

Figure 3

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(A) Kymographs of eGFP-labeled PICH (~50 pM) on DNA molecules at medium tension (25 pN) show that under low salt conditions (25mM NaCl, top), interactions last much longer than in high salt (100 mM, bottom). See also Movies S1 and S2. (B/C) Three independent traces of PICH-eGFP interactions on DNA using high (B) or low (C) salt reaction conditions to demonstrate typical translocation characteristics of PICH. In all cases, the displacement scales linearly with time, as expected for an ATP-dependent translocase. Note the frequent reversal of translocation direction, which it is especially prominent under low salt conditions. Occasional pausing is also apparent. (D) The histograms of observed translocation speeds display a Gaussian distribution in both low and high salt conditions. (E) The cumulative distribution of switch times (i.e., the time that elapses between each reversal of translocation direction) determined under low salt conditions is well fitted by an exponential decay with a characteristic time of ~39 ± 3 s. (F) Because in low salt the switch time is much shorter than the average interaction time, PICH motion resembles random diffusion. See also Figures S6 and S7.

Visualization of PICH translocation in real time

Most PICH-DNA interaction events displayed kymograph lines that deviated from a horizontal course, which could be attributed to the PICH molecules being able to translocate along the DNA (Figure 3A-C). These translocation events were directed and ATP-dependent (Figure S6D), as expected for a SNF2 family ATPase. From velocity histograms, we deduced an average translocation speed of 9.8 ± 0.2 nm/s (approximately 30 bp/s) at 100 mM NaCl (Figure 3D). In low salt buffer (25 mM NaCl), the translocation rate was marginally lower (7.2 ± 0.2 nm/s).

Interestingly, we observed that the direction of protein translocation switched spontaneously, an effect that was more obvious in reactions conducted under low salt conditions (Figure 3C). Moreover, this switching behaviour was observed using various different protein concentrations, including a very low concentration (5 pM), indicating that switching occurs for PICH molecules that are very likely to be in a monomeric state (Figure S7). After switching, neither the speed nor average distance of translocation changed significantly. The average time that elapsed from one directional switching episode to the next was well described by a mono-exponential decay, yielding a switch time of ~40 s (Figure 3E). This corresponds to a translocation processivity of ~800 bp. The switching time was largely independent of the salt concentration: at high salt, we obtained a similar value (>50 s, see Supplemental Information). Thus the observed switching is very unlikely to be caused by protein hopping. Interestingly, the kymographs of long duration events resemble random translocation due to 1D diffusion, even though the translocation is driven by a molecular motor (Figure 3F). This is of note because it is generally assumed that random motion is driven by diffusion (i.e. thermal energy).

Dependence of PICH binding on DNA tension

The use of optical tweezers permits control of the degree of tension that is applied to the DNA. This is of particular interest in the case of PICH because it binds to DNA strands (anaphase bridges) that are presumed to be under tension as a result of their attachment to sister chromatids being pulled toward opposite poles of the mitotic spindle. To test the possibility that PICH might recognize stretched DNA, we recorded the binding of PICH to DNA while increasing the force applied to the DNA from 1 pN to ~55 pN, the maximum force at which the DNA double helix remains stable (Gross et al., 2011; van Mameren et al., 2009b) (Figure 4A/B and Movie S3). Applying higher tensions significantly increased the affinity of PICH for DNA through a decrease in the protein off-rate (Figure 4C). A force increase from 1 pN to 30 pN led to a 10-fold increase of the interaction time of PICH with the DNA (from 1.5 s to 14.5 s). At higher forces (>35 pN), the interaction time reached a maximum and then declined slightly (Figure 4D). Hence, our data indicate that the efficiency of binding to DNA by PICH is strongly enhanced by the application of stretching forces to the DNA up to a level of 35 pN.

Figure 4. PICH responds to DNA stretching.

Figure 4

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(A/B) Kymographs of PICH-EGFP interactions at different DNA tension. The red dotted line denotes where a change of the tension was applied, the respective value of which is given in the upper section in yellow. Note that only the duration, but not the frequency, of interactions changes significantly with DNA tension. See also Movie S3. (C) Cumulative distributions of the interaction time on DNA for PICH-eGFP at different forces follow a mono-exponential decay. (D) The interaction time of PICH on DNA increases by ~10-fold as the force is increased from 1 to 30 pN, and displays a mono-exponential tension dependence (red triangles). At higher tensions, >30 pN, the interaction decreases slightly again. For comparison, the interactions at equivalent PICH-eGFP concentrations after addition of a 15-fold excess of non-labeled PICH (see Figure 6 below) follow the same trend, albeit with an about 3-fold longer interaction time (black circles).

Next, we asked if the apparently novel properties of PICH, most notably the ability to respond to the level of DNA tension, might be a common property of the Rad54 subgroup of SNF2 family proteins. To investigate this, we undertook a direct comparison between eGFP-labeled PICH and hRAD54 (Figures 5A-C). We found that the DNA binding properties of PICH and hRAD54 were strikingly different. For example, under ‘physiological’ buffer conditions, where PICH has a high affinity for binding dsDNA, we could not detect hRAD54:DNA interactions. In agreement with previous data, however, binding of hRAD54 to dsDNA could be detected in buffer containing a very low salt concentration (Amitani et al., 2006; Bianco et al., 2007). Under these conditions, we observed not only ATP-dependent translocation of hRAD54, but also ATP-independent diffusion on the DNA. This latter property also distinguishes hRAD54 from PICH, because PICH remains stationary in the absence of ATP. Moreover, we found that the interaction time of hRAD54 with the DNA is at least an order of magnitude longer than that for PICH under these buffer conditions. Finally, and most significantly, we did not observe any changes in the binding to or dissociation from the DNA of hRAD54 following changes in the degree of DNA tension (Figure 5C). We conclude, therefore, that PICH possesses features unlike those of a very closely related SNF2 family member, and has adaptations that are consistent with a role in recognizing and binding to DNA under tension in anaphase.

Figure 5. hRAD54 has different DNA-binding properties from PICH.

Figure 5

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(A) hRAD54-eGFP binding to DNA. Snapshots of hRad54-eGFP binding to DNA in low salt buffer either without ATP (left) or with ATP (right). (B) Comparison of hRAD54 and PICH shows that, in low salt, hRAD54 (Ia, left) displays rapid diffusion without ATP, while PICH (IIa, right) remains static. In the presence of ATP and Mg2+, hRad54 shows a combination of diffusion and directed motion (Ib), whereas PICH translocates solely with a directed motion (IIb). (C) Comparison of PICH (I, left) and hRAD54 (II, right) interactions before (a, upper) and after (b, lower) DNA overstretching demonstrates that only PICH displays a force-dependent binding; interactions of PICH are significantly depleted by overstretching, while hRad54 binding is unaffected.

PICH oligomerization and structural impact of PICH on DNA

Next, we tested if oligomerization might occur at high PICH concentrations. Because of the high affinity of PICH monomers for DNA, it was not possible to track individual PICH molecules at high protein concentrations because the density of fluorescent spots on the DNA would exceed the resolution limit of the instrument (250 nm). We therefore chose an indirect way to demonstrate the occurrence of PICH oligomers at higher concentrations. We analyzed whether the interactions between low concentrations of PICH-eGFP (80 pM) and DNA displayed any change in characteristics upon addition of a 15-fold molar excess of non-fluorescent PICH (Figure 6A). We found that the interaction time of the bound PICH-eGFP increased significantly upon addition of unlabeled PICH (Figure 6B). This not only indicates the occurrence of PICH oligomerization, but also demonstrates that these oligomers bind more stably to DNA. Notably, the PICH oligomers also displayed an ability to translocate. However, the distance translocated was small (<100 nm), most likely because at this high protein density any motion of the PICH oligomers would be obstructed. Importantly, the stabilization of binding under tension-induced stretching seen with the monomers was also seen with the oligomers (Figure 6C).

Figure 6. Effects of PICH on the physical properties of DNA.

Figure 6

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(A/B) Kymographs of PICH-eGFP (80 pM) interactions with DNA, either before (A) or after (B) addition of a 15-fold molar excess of non-labeled PICH. Note the clear increase of the interaction time at higher PICH concentrations. (C) PICH-eGFP together with non-labeled PICH display a similar force dependence of the interaction time to PICH-eGFP alone, such that, up to 30 pN, higher DNA tension results in a longer interaction time. (D) The force-extension curve of a PICH-coated DNA (red) shows that, compared to normal DNA (black), there is, an earlier force increase combined with a shift to longer extension beyond 30 pN. This effect is much more pronounced in the presence of ATP (blue). Furthermore, the overstretching plateau of normal DNA (black) at >60 pN force has given way to a strong force-increasing regime, showing that PICH binding stabilizes DNA against force-induced melting.

Since UFBs in cells are densely coated with PICH, we also recorded force-extension curves using PICH-saturated DNA (Figure 6D). We deduced the physical properties of the coated DNA that was being exposed to low to moderate levels of force (1-30 pN) by fitting the observed data with the extended worm-like chain (Odijk, 1995). We found that, compared to uncoated DNA, the persistence length of the PICH-coated DNA was significantly reduced, while the contour length was increased (Supplemental Information). This indicates that PICH binding results in a simultaneous bending and lengthening of the DNA duplex. Similar effects were seen for all ATP-binding variants (PICH, PICH-eGFP and eGFP-PICH; Figure S5C). Interestingly, we found that the impact of PICH on DNA conformation strongly depended on whether nucleotides were present in the reaction. In the presence of ATP, DNA bending and lengthening was significantly more pronounced than it is in the absence of ATP (Figure 6D). Another notable feature of the PICH-coated DNA was the disappearance of the overstretching plateau at high forces (~65 pN), which for naked DNA denotes force-induced strand separation (i.e. melting) (Gross et al., 2011; van Mameren et al., 2009a). Instead, with PICH bound to DNA, the force-induced overstretching increased monotonously with the extension, indicating that PICH stabilizes the DNA double helix.

DISCUSSION

Biochemical and biophysical characteristics of PICH

We have utilized both ensemble and single-molecule techniques to investigate the biochemical and biophysical properties of the human PICH protein, in order to gain insight into its biological role in mitosis. Broadly speaking, our data show that PICH possesses the expected biochemical properties of a SNF2 family member (Enemark and Joshua-Tor, 2008; Pazin and Kadonaga, 1997) in being a DNA-dependent ATPase and an ATP-dependent translocase, but lacking helicase activity. Nevertheless, from comparisons of our data on PICH with those in the literature, together with our own studies on hRad54, it is clear that PICH differs significantly in its properties even from close family members such as hRAD54 or yeast Rad54 and Rdh54 (yRad54/yRdh54). For example, yRad54 is capable of migrating on dsDNA at 300 bp/s over distances >10 kb (Amitani et al., 2006; Bianco et al., 2007; Ceballos and Heyer, 2011; Mazin et al., 2010; Nimonkar et al., 2007; Prasad et al., 2007; Tan et al., 2003). The mechanism by which yRad54 achieves such an impressively high speed and processivity is not fully elucidated, but likely requires the enzyme to adopt a multimeric (and possibly ring-like) assembly on the DNA. In contrast, our data show that PICH differs from yRad54 in several respects: (i) PICH is a slower and much less processive translocase. (ii) PICH displays a four orders of magnitude higher affinity for dsDNA than does yRad54 (Ceballos and Heyer, 2011). (iii) The duration of DNA binding by PICH is more transient (0.1 s−1, under physiological conditions). These distinct properties likely reflect differences in the assembly state of PICH and yRad54, because our data indicate that PICH is capable of translocation even when in a monomeric assembly state. We did obtain evidence for oligomerization of PICH at elevated concentrations; however, we found no indication that PICH oligomers display fundamentally different enzymatic properties from the monomeric form.

One notable feature of PICH is that it is able to spontaneously reverse its direction of translocation. This feature is also observed for some Rad54 family proteins. In those cases, it is usually attributed to different protomers of the oligomer making contact with the DNA in turn, which for a monomer such as PICH would not be possible. We considered the possibility that the observed switching might originate from PICH dimers or oligomers in the preparation, which might be ‘invisible’ due to there being a substantial fraction of non-fluorescent eGFP moieties. However, we obtained no indication for any protein concentration dependence of the switching time, and hence we believe this possibility to be unlikely. It might be that monomeric PICH has two independent DNA binding sites, and that switching is caused by the dissociation of DNA from one site and subsequent rebinding to the other site after reorientation of the protein. Because of the frequent switching of direction, the translocation distance between switching events is relatively low for PICH. Hence, over longer time scales, the net translocation distance would scale with the square root of time, which is identical to 1D diffusion. However, for an enzyme such as PICH that is ‘designed’ to coat anaphase bridge DNA at high density, translocation over long distances would not be expected to be a requirement. Despite the similarities of PICH translocation with 1D diffusion, there is one important difference: PICH consumes energy while moving, which allows for force generation with respect to the DNA and which in turn could be important if PICH needs to remove obstacles from the DNA (see discussion below).

The force-extension curves of PICH-coated DNA showed that PICH binding results in a number of changes to the structure of the DNA. PICH binding generates DNA bending, an increase in the DNA contour length and a stabilization of the DNA against unwinding due to overstretching. These findings are corroborated by the force dependence of the interaction time: at levels of tension below 30 pN, PICH binding to DNA is stabilized because the lengthening of the DNA is facilitated by the application of higher forces. In particular, the exponential force dependence can be used to calculate that a single PICH monomer lengthens the DNA duplex by 0.32 ±0.08 nm. At the same time, the bending induced by the protein is negatively affected by the application of stretching forces, such that at forces above 30 pN the corresponding destabilization of the PICH-DNA interaction outweighs the effect of DNA lengthening. This tension sensitivity is not a general feature of all SNF2 family proteins, as evident from our demonstration that hRAD54 binding to DNA is neither increased nor decreased by the application of stretching forces to the DNA.

Does PICH remodel nucleosomes?

Recently, it was proposed that the role of PICH might be to remodel chromatin on UFBs (Ke et al., 2011). While this proposal is consistent with the fact that UFBs do not seem to be chromatinized, the reported remodeling activity was very weak; indeed, it was several orders of magnitude less efficient than that seen with Chd1, a bona fide remodeler of the same family (Ke et al., 2011). We could not reproduce the findings of Ke et al. using our PICH and human nucleosomes, despite undertaking analysis of several different PICH preparations. Moreover, we also failed to see remodeling using the centromeric human H3 histone CENP-A, which we tested because of the possibility that PICH might show specificity for centromeric nucleosomes. Additional evidence that PICH is unlikely to expel nucleosomes by itself was discussed recently (Kaulich et al., 2012).

A new model for the function of PICH in the maintenance of genome stability

We propose a model (Figure 7) for the role of PICH in mitosis that combines our new findings on the biophysical properties of PICH with current knowledge on the characteristics of chromatin being subjected to stretching forces (Chien and van Noort, 2009). Because the binding of PICH to DNA is initiated by charge interactions, it might be expected that PICH has a low affinity for normal chromatin, where much of the phosphate backbone is shielded by nucleosomes. If, however, during early anaphase, an attempt is made to segregate sister chromatids that still contain catenated DNA, there would be a build-up of force initially near the point of entanglement. This would immediately affect the chromatin structure, because nucleosomes are very sensitive force sensors and only a few pN of force are needed to initiate unwrapping of individual nucleosomes to the point that linker DNA (typically 20-60 bp long) is exposed to solution (Kruithof et al., 2009). Based on our demonstration that PICH has a remarkably high affinity for dsDNA molecules as short as 20 bp, it is reasonable to assume that PICH monomers would bind rapidly to any regions of bare linker DNA exposed by tension-induced nucleosome unwrapping. The DNA exposed by nucleosome eviction would also be under tension, which would result in a stabilization of the bound PICH to the DNA. Since tension weakens nucleosome binding, it also lowers the energy barrier that a translocase enzyme like PICH would need in order to effect nucleosome eviction. Hence, although we have no experimental evidence for this, we certainly do not rule out the possibility that PICH might possess some ability in vivo to facilitate nucleosome eviction. In fact, a force-dependent nucleosome eviction activity function for PICH would explain why we observed no activity on properly assembled nucleosome samples in vitro. It should be noted, however, that our model does not rely on the existence of any force-dependent nucleosome eviction by PICH, since the affinity of PICH for UFBs will depend on the combined effect of the sensitivity of the nucleosome (weakening with increasing tension) and of PICH binding to DNA (stabilizing with increasing tension). Hence, even without active nucleosome eviction, the formation of a PICH coated UFB would be favored. This is in agreement with results (Baumann et al., 2007) showing that an ATPase-dead variant of PICH can still localize to UFBs, even though this variant lacks the ability to translocate and actively evict nucleosomes. Once bound to the UFB, we propose that PICH plays two additional roles. First, it acts to stabilize the DNA from stretching-induced DNA denaturation. This would be of particular importance if the DNA contains nicks or single-stranded regions that would be prone to force-induced unraveling of the complementary strands. Second, PICH recruits other proteins to the UFBs, including the BTRR complex (Baumann et al., 2007; Chan and Hickson, 2011; Chan et al., 2007; Ke et al., 2011; Ying and Hickson, 2011).

Figure 7.

Figure 7

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Proposed model for the action of PICH on UFB formation and resolution. (I) At the start of mitosis, the chromatin is still in the 30 nm fibre configuration. Since the nucleosomes at least partially shield the phosphate backbone of the DNA, PICH is not able to bind stably. (II) Once sister chromatid disjunction is triggered, there is a build-up of tension leading to DNA extension (denoted by the upward facing arrows), which causes fibre elongation, without exposing bare DNA. (III) Beyond a certain force threshold, the area close to the entanglement is forced to extend further into a beads-on-a-string configuration; PICH will accordingly bind to the exposed linker DNA (~50 bp) and is then able to translocate in either direction. (IV) Aided by the tension on the corresponding DNA section, PICH may be able to assist in overcoming the energy barrier for the unwrapping of nucleosomes with a ‘weakened grip’ on the DNA adjacent to the exposed section. This would expose more free DNA, thus recruiting more PICH mono- and oligomers to the exposed region of DNA. (V) Nucleosomes are expelled from the DNA, which becomes coated with PICH. This has the dual effect of stabilizing the DNA against stretching-induced strand unwinding, and allowing the DNA tension to be maintained at a constant level, as denoted by the horizontal arrows. Finally, PICH recruits the BTRR complex and possibly other DNA metabolising enzyme to permit the disentanglement of the intertwined DNA that had prevented normal disjunction in the first place.

In summary, our new model posits that the interplay between force-induced nucleosome unwrapping and PICH binding to DNA could be the central tension sensing mechanism that starts the chain of catalytic events that leads to the resolution of UFBs during mitosis, a reaction that we propose is catalyzed either by topoisomerase II or by the BTRR complex.

EXPERIMENTAL PROCEDURES

Generation of constructs and DNA substrates

Details of the construction of PICH expression plasmids and DNA triplexes can be found in the Supplemental Information.

Recombinant PICH protein expression and purification

In order to characterise the biochemical and biophysical properties of PICH, we purified four recombinant versions of the protein from baculovirus-infected insect cells. Two were hexa-histidine tagged, full-length PICH proteins (hereafter termed ‘native PICH’) that were not fused to GFP. Details of the purification of these proteins can be found in the Supplemental Information.

ATPase assays

The ATPase activity of PICH was assayed using a commercial kit (Innova Biosciences) according to the manufacturer’s instructions. Where indicated, reactions contained 500 ng of a specified DNA co-factor.

DNA binding assays

Electrophoretic mobility shift assays (EMSAs) for analyzing the binding of purified PICH or PICH-K128A protein to DNA were conducted as described in the Supplemental Information.

Translocase and branch migration assays

Details of the PICH migration assays can be found in the Supplemental Information.

Nucleosome remodeling assays

Recombinant human histone proteins were expressed and purified as described previously (Flaus et al., 2004) except for human CENP-A, which was expressed as a soluble tetramer with histone H4 as described by Black et al. (Black et al., 2004). Yeast RSC was expressed and purified as described by Ferreira et al. (Ferreira et al., 2007). Nucleosomes were assembled using standard salt-gradient dialysis methods using recombinant human histones and PCR-generated DNA fragments. The Cy5 or Cy3 labelled PCR fragments contained the 601 or MMTV NucA nucleosome positioning sequences in the centre (47w47, 54A54) or at the end (0w47). Nucleosome sliding assays were performed in 50 mM NaCl, 50 mM Tris (pH 7.5), and 3 mM MgCl2 plus or minus 1 mM ATP, as indicated in the Figure. The reactions were incubated at 37°C with the amount of RSC or PICH specified in the Figure. Reactions were stopped after 30 min by addition of 1 μg of plasmid DNA and sucrose to 5% (w/v) before electrophoresis on 0.2xTris-Borate-EDTA (TBE) 5% native polyacrylamide gels. The gels were scanned for the Cy-dyes signal using a PhosphorImager.

Single molecule assays

Single molecule experiments were carried out in a set-up combining fluorescence microscopy with double optical tweezers (Candelli et al., 2011; Farge et al., 2012; van Mameren et al., 2009b), as described in the Supplemental Information.

Supplementary Material

Movie S1

Movie S1: Fluorescence movie of 50 nM PICH-eGFP DNA-binding and –unbinding at medium tension (30 pN) in high salt buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 2 mM MgCl2, 2 mM ATP). Note that the movie is speeded up by 35-fold (from 1 to 35 Hz) compared to real time, and that the interactions are much shorter than seen in low salt buffer (Movie S2). The bead diameter can be used as scalebar (3.3 μm diameter).

Download video file (8.7MB, mp4)
Movie S2

Movie S2: Fluorescence movie of PICH-eGFP DNA-binding and –unbinding at medium tension (30 pN) in low salt buffer (20 mM Tris-HCl pH 7.5, 25 mM NaCl, 2 mM MgCl2, 2 mM ATP). Note that the movie is speeded up by 35-fold (from 0.4 to 14 Hz) in order to render the slow PICH translocation more easily discernible. The bead diameter can be used as scalebar (3.3 μm diameter).

Download video file (9.7MB, mp4)
Movie S3

Movie S3: Fluorescence movie of PICH-eGFP DNA-binding and –unbinding in high salt buffer (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 2 mM MgCl2, 2 mM ATP) at different DNA tensions; DNA tension is increased from the initial 2 pN stepwise to 16 (10 s), 30 (23), 47 (34) and finally 57 pN (47 s) by moving the right bead. Note that the movie is speeded up by 25-fold (from 1 to 25 Hz) compared to real time. The bead diameter can be used as scalebar (3.3 μm diameter).

Download video file (14.1MB, mp4)
Supplementary Material

ACKNOWLEDGEMENTS

We would like to thank members of the Hickson, Nigg, Peterman and Wuite groups for helpful discussions. Work in the authors’ laboratories is supported by Cancer Research (UK), The Nordea Foundation (Denmark), The Association for International Cancer Research (UK), The Danish Cancer Society, The Novo Nordisk Foundation (Denmark), LaserLab Europe, and the University of Basel (Switzerland). This work is also part of a research programme of the Foundation for Fundamental Research on Matter (FOM), which is part of the Netherlands Organisation for Scientific Research (NWO). Finally, we acknowledge support by a VICI of now, as well as an ERC starting grant.

REFERENCES

  1. Amitani I, Baskin RJ, Kowalczykowski SC. Visualization of Rad54, a chromatin remodeling protein, translocating on single DNA molecules. Molecular Cell. 2006;23:143–148. doi: 10.1016/j.molcel.2006.05.009. [DOI] [PubMed] [Google Scholar]
  2. Baumann C, Korner R, Hofmann K, Nigg EA. PICH, a centromere-associated SNF2 family ATPase, is regulated by Plk1 and required for the spindle checkpoint. Cell. 2007;128:101–114. doi: 10.1016/j.cell.2006.11.041. [DOI] [PubMed] [Google Scholar]
  3. Bianco PR, Bradfield JJ, Castanza LR, Donnelly AN. Rad54 oligomers translocate and cross-bridge double-stranded DNA to stimulate synapsis. Journal of Molecular Biology. 2007;374:618–640. doi: 10.1016/j.jmb.2007.09.052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Black BE, Foltz DR, Chakravarthy S, Luger K, Woods VL, Jr., Cleveland DW. Structural determinants for generating centromeric chromatin. Nature. 2004;430:578–582. doi: 10.1038/nature02766. [DOI] [PubMed] [Google Scholar]
  5. Cairns BR, Lorch Y, Li Y, Zhang M, Lacomis L, Erdjument-Bromage H, Tempst P, Du J, Laurent B, Kornberg RD. RSC, an essential, abundant chromatin-remodeling complex. Cell. 1996;87:1249–1260. doi: 10.1016/s0092-8674(00)81820-6. [DOI] [PubMed] [Google Scholar]
  6. Candelli A, Wuite GJ, Peterman EJ. Combining optical trapping, fluorescence microscopy and micro-fluidics for single molecule studies of DNA-protein interactions. Phys Chem Chem Phys. 2011;13:7263–7272. doi: 10.1039/c0cp02844d. [DOI] [PubMed] [Google Scholar]
  7. Ceballos SJ, Heyer WD. Functions of the Snf2/Swi2 family Rad54 motor protein in homologous recombination. Biochim Biophys Acta. 2011;1809:509–523. doi: 10.1016/j.bbagrm.2011.06.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chan KL, Hickson ID. New insights into the formation and resolution of ultra-fine anaphase bridges. Semin Cell Dev Biol. 2011;22:906–912. doi: 10.1016/j.semcdb.2011.07.001. [DOI] [PubMed] [Google Scholar]
  9. Chan KL, North PS, Hickson ID. BLM is required for faithful chromosome segregation and its localization defines a class of ultrafine anaphase bridges. Embo J. 2007;26:3397–3409. doi: 10.1038/sj.emboj.7601777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chan KL, Palmai-Pallag T, Ying S, Hickson ID. Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat Cell Biol. 2009;11:753–760. doi: 10.1038/ncb1882. [DOI] [PubMed] [Google Scholar]
  11. Chaurasiya KR, Paramanathan T, McCauley MJ, Williams MC. Biophysical characterization of DNA binding from single molecule force measurements. Phys Life Rev. 2010;7:299–341. doi: 10.1016/j.plrev.2010.06.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chien FT, van Noort J. 10 years of tension on chromatin: results from single molecule force spectroscopy. Curr Pharm Biotechnol. 2009;10:474–485. doi: 10.2174/138920109788922128. [DOI] [PubMed] [Google Scholar]
  13. Chu WK, Hickson ID. RecQ helicases: multifunctional genome caretakers. Nat Rev Cancer. 2009;9:644–654. doi: 10.1038/nrc2682. [DOI] [PubMed] [Google Scholar]
  14. Durkin SG, Glover TW. Chromosome fragile sites. Annu Rev Genet. 2007;41:169–192. doi: 10.1146/annurev.genet.41.042007.165900. [DOI] [PubMed] [Google Scholar]
  15. Durr H, Korner C, Muller M, Hickmann V, Hopfner KP. X-ray structures of the Sulfolobus solfataricus SWI2/SNF2 ATPase core and its complex with DNA. Cell. 2005;121:363–373. doi: 10.1016/j.cell.2005.03.026. [DOI] [PubMed] [Google Scholar]
  16. Enemark EJ, Joshua-Tor L. On helicases and other motor proteins. Curr Opin Struct Biol. 2008;18:243–257. doi: 10.1016/j.sbi.2008.01.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Farge G, Laurens N, Broekmans OD, van den Wildenberg SM, Dekker LC, Gaspari M, Gustafsson CM, Peterman EJ, Falkenberg M, Wuite GJ. Protein sliding and DNA denaturation are essential for DNA organization by human mitochondrial transcription factor A. Nat Commun. 2012;3:1013. doi: 10.1038/ncomms2001. [DOI] [PubMed] [Google Scholar]
  18. Ferreira H, Flaus A, Owen-Hughes T. Histone modifications influence the action of Snf2 family remodelling enzymes by different mechanisms. Journal of molecular biology. 2007;374:563–579. doi: 10.1016/j.jmb.2007.09.059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Flaus A, Rencurel C, Ferreira H, Wiechens N, Owen-Hughes T. Sin mutations alter inherent nucleosome mobility. Embo J. 2004;23:343–353. doi: 10.1038/sj.emboj.7600047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. German J. Bloom syndrome: a mendelian prototype of somatic mutational disease. Medicine (Baltimore) 1993;72:393–406. [PubMed] [Google Scholar]
  21. Gisselsson D. Chromosome instability in cancer: how, when, and why? Adv Cancer Res. 2003;87:1–29. doi: 10.1016/s0065-230x(03)87164-6. [DOI] [PubMed] [Google Scholar]
  22. Gross P, Farge G, Peterman EJ, Wuite GJ. Combining optical tweezers, single-molecule fluorescence microscopy, and microfluidics for studies of DNA-protein interactions. Methods Enzymol. 2010;475:427–453. doi: 10.1016/S0076-6879(10)75017-5. [DOI] [PubMed] [Google Scholar]
  23. Gross P, Laurens N, Oddershede LB, Bockelmann U, Peterman EJG, Wuite GJL. Quantifying how DNA stretches, melts and changes twist under tension. Nature Physics. 2011;7:731–736. [Google Scholar]
  24. Hubner NC, Wang LH, Kaulich M, Descombes P, Poser I, Nigg EA. Re-examination of siRNA specificity questions role of PICH and Tao1 in the spindle checkpoint and identifies Mad2 as a sensitive target for small RNAs. Chromosoma. 2010;119:149–165. doi: 10.1007/s00412-009-0244-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kaulich M, Cubizolles F, Nigg EA. On the regulation, function, and localization of the DNA-dependent ATPase PICH. Chromosoma. 2012;121:395–408. doi: 10.1007/s00412-012-0370-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ke Y, Huh JW, Warrington R, Li B, Wu N, Leng M, Zhang J, Ball HL, Li B, Yu H. PICH and BLM limit histone association with anaphase centromeric DNA threads and promote their resolution. Embo J. 2011;30:3309–3321. doi: 10.1038/emboj.2011.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kruithof M, Chien FT, Routh A, Logie C, Rhodes D, van Noort J. Single-molecule force spectroscopy reveals a highly compliant helical folding for the 30-nm chromatin fiber. Nat Struct Mol Biol. 2009;16:534–540. doi: 10.1038/nsmb.1590. [DOI] [PubMed] [Google Scholar]
  28. Kurasawa Y, Yu-Lee LY. PICH and cotargeted Plk1 coordinately maintain prometaphase chromosome arm architecture. Mol Biol Cell. 2010;21:1188–1199. doi: 10.1091/mbc.E09-11-0950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Leng M, Bessuso D, Jung SY, Wang Y, Qin J. Targeting Plk1 to chromosome arms and regulating chromosome compaction by the PICH ATPase. Cell Cycle. 2008;7:1480–1489. doi: 10.4161/cc.7.10.5951. [DOI] [PubMed] [Google Scholar]
  30. Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature. 1998;396:643–649. doi: 10.1038/25292. [DOI] [PubMed] [Google Scholar]
  31. Mazin AV, Mazina OM, Bugreev DV, Rossi MJ. Rad54, the motor of homologous recombination. DNA Repair (Amst) 2010;9:286–302. doi: 10.1016/j.dnarep.2009.12.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Naim V, Rosselli F. The FANC pathway and BLM collaborate during mitosis to prevent micro-nucleation and chromosome abnormalities. Nat Cell Biol. 2009;11:761–768. doi: 10.1038/ncb1883. [DOI] [PubMed] [Google Scholar]
  33. Nimonkar AV, Amitani I, Baskin RJ, Kowalczykowski SC. Single molecule imaging of Tid1/Rdh54, a Rad54 homolog that translocates on duplex DNA and can disrupt joint molecules. The Journal of biological chemistry. 2007;282:30776–30784. doi: 10.1074/jbc.M704767200. [DOI] [PubMed] [Google Scholar]
  34. Odijk T. Stiff Chains and Filaments under Tension. Macromolecules. 1995;28:7016–7018. [Google Scholar]
  35. Pazin MJ, Kadonaga JT. SWI2/SNF2 and related proteins: ATP-driven motors that disrupt protein-DNA interactions? Cell. 1997;88:737–740. doi: 10.1016/s0092-8674(00)81918-2. [DOI] [PubMed] [Google Scholar]
  36. Prasad TK, Robertson RB, Visnapuu ML, Chi P, Sung P, Greene EC. A DNA-translocating Snf2 molecular motor: Saccharomyces cerevisiae Rdh54 displays processive translocation and extrudes DNA loops. Journal of molecular biology. 2007;369:940–953. doi: 10.1016/j.jmb.2007.04.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tan TL, Kanaar R, Wyman C. Rad54, a Jack of all trades in homologous recombination. DNA Repair (Amst) 2003;2:787–794. doi: 10.1016/s1568-7864(03)00070-3. [DOI] [PubMed] [Google Scholar]
  38. van den Wildenberg SMJL, Bollen YJM, Peterman EJG. How to quantify protein diffusion in the bacterial membrane. Biopolymers. 2011;95:312–321. doi: 10.1002/bip.21585. [DOI] [PubMed] [Google Scholar]
  39. van Mameren J, Gross P, Farge G, Hooijman P, Modesti M, Falkenberg M, Wuite GJ, Peterman EJ. Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging. Proc Natl Acad Sci U S A. 2009a;106:18231–18236. doi: 10.1073/pnas.0904322106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. van Mameren J, Modesti M, Kanaar R, Wyman C, Peterman EJ, Wuite GJ. Counting RAD51 proteins disassembling from nucleoprotein filaments under tension. Nature. 2009b;457:745–748. doi: 10.1038/nature07581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wang LH, Mayer B, Stemmann O, Nigg EA. Centromere DNA decatenation depends on cohesin removal and is required for mammalian cell division. J Cell Sci. 2010;123:806–813. doi: 10.1242/jcs.058255. [DOI] [PubMed] [Google Scholar]
  42. Wang LH, Schwarzbraun T, Speicher MR, Nigg EA. Persistence of DNA threads in human anaphase cells suggests late completion of sister chromatid decatenation. Chromosoma. 2008;117:123–135. doi: 10.1007/s00412-007-0131-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Whitby MC, Lloyd RG. Targeting Holliday junctions by the RecG branch migration protein of Escherichia coli. The Journal of biological chemistry. 1998;273:19729–19739. doi: 10.1074/jbc.273.31.19729. [DOI] [PubMed] [Google Scholar]
  44. Ying S, Hickson ID. Fanconi anaemia proteins are associated with sister chromatid bridging in mitosis. Int J Hematol. 2011;93:440–445. doi: 10.1007/s12185-011-0818-7. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Movie S1

Movie S1: Fluorescence movie of 50 nM PICH-eGFP DNA-binding and –unbinding at medium tension (30 pN) in high salt buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 2 mM MgCl2, 2 mM ATP). Note that the movie is speeded up by 35-fold (from 1 to 35 Hz) compared to real time, and that the interactions are much shorter than seen in low salt buffer (Movie S2). The bead diameter can be used as scalebar (3.3 μm diameter).

Download video file (8.7MB, mp4)
Movie S2

Movie S2: Fluorescence movie of PICH-eGFP DNA-binding and –unbinding at medium tension (30 pN) in low salt buffer (20 mM Tris-HCl pH 7.5, 25 mM NaCl, 2 mM MgCl2, 2 mM ATP). Note that the movie is speeded up by 35-fold (from 0.4 to 14 Hz) in order to render the slow PICH translocation more easily discernible. The bead diameter can be used as scalebar (3.3 μm diameter).

Download video file (9.7MB, mp4)
Movie S3

Movie S3: Fluorescence movie of PICH-eGFP DNA-binding and –unbinding in high salt buffer (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 2 mM MgCl2, 2 mM ATP) at different DNA tensions; DNA tension is increased from the initial 2 pN stepwise to 16 (10 s), 30 (23), 47 (34) and finally 57 pN (47 s) by moving the right bead. Note that the movie is speeded up by 25-fold (from 1 to 25 Hz) compared to real time. The bead diameter can be used as scalebar (3.3 μm diameter).

Download video file (14.1MB, mp4)
Supplementary Material
NIHMS60325-supplement-01.pdf (1.5MB, pdf)

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