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Title: DNA Dynamics during Early Double-Strand Break Processing Revealed by Non-Intrusive Imaging of Living Cells}
Article:
Abstract:
Chromosome breakage is a major threat to genome integrity. The most accurate way to repair DNA double strand breaks( DSB) is homologous recombination( HR) with an intact copy of the broken locus. Mobility of the broken DNA has been seen to increase during the search for a donor copy. Observing chromosome dynamics during the earlier steps of HR, mainly the resection from DSB ends that generates recombinogenic single strands, requires a visualization system that does not interfere with the process, and is small relative to the few kilobases of DNA that undergo processing. Current visualization tools, based on binding of fluorescent repressor proteins to arrays of specific binding sites, have the major drawback that highly-repeated DNA and lengthy stretches of strongly bound protein can obstruct chromatin function. We have developed a new, non-intrusive method which uses protein oligomerization rather than operator multiplicity to form visible foci. By applying it to HO cleavage of the MAT locus on Saccharomyces cerevisiae chromosome III, we provide the first real-time analysis of resection in single living cells. Monitoring the dynamics of a chromatin locus next to a DSB revealed transient confinement of the damaged chromatin region during the very early steps of resection, consistent with the need to keep DNA ends in contact. Resection in a yku70 mutant began ∼10 min earlier than in wild type, defining this as the period of commitment to homology-dependent repair. Beyond the insights into the dynamics and mechanism of resection, our new DNA-labelling and-targeting method will be widely applicable to fine-scale analysis of genome organization, dynamics and function in normal and pathological contexts.
Introduction:
DNA double strand breaks( DSB) are a major threat to chromosome integrity and cell survival. Cells meet it by launching repair programs consisting of the enzymatic restoration of the DNA and of appropriate chromatin remodelling and checkpoint activation. The exposed DNA ends are protected by the Ku70-Ku80 complex( Ku complex) until a repair pathway is chosen and corresponding proteins recruited. Direct resealing of breaks by non-homologous end joining( NHEJ) is promoted by the Ku complex but is error-prone[1]. The most precise repair pathway is replacement of the broken segment with an intact copy by homologous recombination( HR), a process conserved throughout the three kingdoms of life[2],[3]. HR is initiated by DNA end processing, during which the nucleolytic activity of the Mre11-Rad50- Xrs2/Nbs1 complex( MRX/MRN) and Sae2/CTIP generates short 3′-ssDNA tails that then serve as the substrate for extensive resection by Exo1 exonuclease or Sgs1-Dna2 helicase/endonuclease[4]–[6]. RPA binds to the exposed ssDNA and acts as a recruiting platform to assemble proteins of the recombination apparatus that enables scanning of the genome for the homologous donor. Observation of resection has relied on indirect immunofluorescence of bromodeoxyuridine-labelled DNA[6] in fixed cells or in vivo imaging of RPA, Rad51 and Rad52 proteins that accumulate on ssDNA close to DSBs( for example see[7],[8]). As a result, the role of chromatin mobility in DSB repair has been investigated almost exclusively in relation to the homology search step that follows resection[9]. Diffusive, undirected motion of chromatin is thought to suffice for inter-chromatid HR, at least on the scale of the yeast nucleus. Tracking DSB repair proteins fused to GFP-type peptides in budding yeast suggested that DSBs gather in “repair centres” containing the HR mediator, Rad52[10],[11]. Labelling of chromosomal sites near irreparable DSBs with fluorescent repressor-operator arrays enabled tracking of their migration to the nuclear periphery[9],[12],[13] and observation that their movement was confined within 2 h of cleavage[8]. When the homologue is present but distant, as in yeast diploid G1 cells, mobilization is needed for pairing[14],[15]. Changes in chromatin mobility accompanying resection itself have received little attention. Assessing DSB processing in single cells demands that we can distinguish cells that have incurred a break and are resecting from those that have not. We therefore sought to identify cells undergoing resection by monitoring loss of a fluorescent tag inserted immediately adjacent to a DSB site. Fluorescent operator/repressor systems( FROS) available for tagging genomic loci in eucaryotes[16]–[19] were not suitable here. The repetitive nature and large size of the operator arrays can alter short-range DNA processes such as gene domain structure, intragenic looping or DNA maintenance, and can also provoke disruptive recombinational events. In addition, tightly bound LacI and TetR repressors can create fragile sites and constitute a barrier of unknown penetrability to DNA processing enzymes[20]–[23]. We have developed an alternative DNA labelling system that circumvents these drawbacks. It has enabled us to identify budding yeast cells that undergo resection following a single HO endonuclease cut. Limiting our analysis to these cells permitted realistic calculation of DNA end resection dynamics as well as measurement of the time taken to commit to HR. In addition, use of the new tool has led to discovery of a distinct phase of confinement of chromatin neighbouring the DSB early in the resection period.
Discussion:
A new picture of the early phase of HR emerges from our findings. While numerous prior studies have identified key players in resection[40]–[46], and have outlined their successive actions and related changes in chromatin mobility[5],[33],[47], the early steps of repair following specific double-strand breakage have not been analysed at the level of single cells. One reason is the inability to directly observe the act of cleavage and the onset of resection in vivo. Despite the general utility of LacI/O- and TetR/O-based FROS in monitoring chromatin positioning and dynamics, the large size of these complexes precludes analysis of the few kilobases of resected DNA, making them inappropriate for quantitative assessment of resection. An alternative approach used to analyze HR is visualization of fluorescently-tagged proteins known to bind to the resected DNA, such as RPA, Rad51 and Rad52[48],[49], but these suffer from some loss of normal function due to the fused fluorescent peptide and intervene only at later stages or after resection is complete. We discovered a severe reduction in mobility of the broken chromosome ends that accompanies the initial phase of resection. The constrained DNA dynamics were not observed in earlier studies which focused on later stages of HR. Loading of repair proteins probably contributes to the rapid reduction in mobility, but cannot by itself account for the significant change in the observed diffusion coefficient. DSB ends may also interact with nuclear structures, adjacent chromatin domains or modified chromatin, possibly reflecting a need to prevent loss of contact between DNA ends prior to homology search. This would represent a security measure, as noted also by Soutoglou et al.[50]. Alternatively, the exposed ends might be held in place by a specific bridge, a role suggested for human and yeast Mre11[51],[52]. A further alternative, that reduced dynamics might be due to an attachment to heterochromatin structures usually found near the nuclear envelope, appears unlikely in view of the retention of the cleaved MAT locus near the nuclear center[37]. Whatever its mechanism, the confinement of the broken ends is transient, suggesting that once the resected ends are processed and the recombination machinery is loaded, the ends now engaged in donor search resume normal chromatin motion. Technical limitations have prevented dissection of early steps in DSB repair. The unavoidably poor synchronization of induced endonuclease( HO or I-SceI) cleavage causes a relatively wide spread in the time of initiation of resection throughout the population. The earliest assays of cleavage have typically been made 30–60 minutes post-induction, but cleavage can still be under way at 4 h, such that the cleavage rate curve is a composite of many temporally dispersed individual resections, each of which might proceed faster than the apparent rate estimated using molecular biological techniques. Our analysis of individual resecting cells shows that this is indeed the case. Thus, our analysis enabled us to determine the time from cleavage to commitment to the HR repair pathway by measuring the difference between wt and yku mutant cells in the time of disappearance of the first fluorescent marker, close to the DSB. During this 10 min period the Ku complex and other proteins intervene to promote ligation by NHEJ( by the Ku complex;[53]), to block resection after its initiation by MRX-Sae2 or Exo1( by Rad9;[54]) and to activate chromatin remodelling[55]). Their activities prepare the DSB ends for repair by HR, NHEJ or other pathways. Focussing analysis on individual cells also allowed us to determine resection speed. The speed we find for the initial resection, which includes both processing by MRX and the first ∼1 kb of resection proper, is comparable to that reported previously. It is unaffected by removal of the HMR and HML recombination target sites. Resection did not extend as far as the INT2 locus, inserted 4. 6 kb from INT1, presumably because assembly of the homology search apparatus curtailed it. In the absence of yKu however, the initial phase was 3–4-fold faster than when measured in wt. Elimination of the decision phase delay rendered the observed speed, ∼300 nt/min, an accurate estimate of the combined nucleolytic activities that resect the first ∼1000 nt, although we cannot exclude the possibility that regulation of resection is perturbed in the absence of the Ku complex. Resection beyond this point is faster still, averaging nearly 1900 nt/min. In this case it can proceed as far as the distant INT2 locus, possibly because rapid onset of resection in the yku mutant leaves insufficient time for diversion of the resected DNA into homology search and the associated termination of nuclease activity. At least two factors could be responsible for this faster resection rate. Relatively slow MRX-mediated processing is not involved, so that digestion of the INT1–INT2 span results solely from Exo1 activity; and either a natural paucity of nucleosomes or nucleosome removal due to DSB-induced triggering of histone modification and check-point pathways might allow Exo1 obstacle-free progress along the MAT locus DNA. The comparable resection rates seen in the presence and absence of INT1 demonstrate that the ParB proteins themselves do not constitute a barrier to progress of the resecting nuclease. The fast rate is also comparable to that of in vitro Exo1 nucleolysis, suggesting that efficient removal of nucleosomes and other bound proteins facilitates Exo1 progress[56],[57]. An unexpected finding was the marked preference for Exo1 as the resection exonuclease, at least in our experimental set up. Previous studies suggested the Sgs1-Dna2 helicase-nuclease pair as an alternative to Exo1 during the fast, extensive resection step[33],[58]. The small fraction of exo1 cells seen to resect suggests that Sgs1-Dna2 may be more dependent than Exo1 on initial processing by MRX-Sae2[34][6]; indeed the MRX complex has been seen to interact directly with Sgs1 in vitro and to stimulate Sgs1-unwinding[58],[59]. Preliminary results from our laboratory( data not shown) indicate that resection dynamics in a mre11 mutant are similar to those in wild type, suggesting that MRX-Sae2 is dispensable for resection from HO breaks at MAT and that as a consequence Sgs1-Dna2 is less readily employed than Exo1 for extensive resection. It is possible in principle that Sgs1-Dna2 progress is more easily blocked by the ParB1-INT1 complex than is Exo1, although the insensitivity to INT1 in wt cells( Fig. 3) makes this an unlikely explanation. Another possible source of the apparent discrepancy between our observation and reported evidence for Sgs1-Dna2 involvement is the extended time-scale of previous resection studies, which resulted from prolonged homology search in the absence of donor loci. In our experiments, with donor loci present, resection was over less than 40 minutes after cleavage induction. Possibly, Sgs1/Dna2 serves as a back-up resection system after prolonged failure to use Exo1. The same might apply to MRX-Sae2 itself, in which case several iterations of resection of small stretches of DNA would be required; such an action could explain the much lower resection rate observed in the exo1 mutant. We further found that a fluorescently labeled genomic locus 3. 4 kb distant from the HO cut site, marked by INT2-GFP, did not disappear in wild type cells. The length of the resected ssDNA tracts in HR-proficient cells has been reported to vary by others. It depended on the availability and location of the homologous template and was correlated with the kinetics of repair. In meiotic cells, the average length of ssDNA formed is 850 nt, whereas 2–4 kb ssDNA tails are formed during mitotic repair between chromosome homologs[60],[61]. We think it likely that the Ku-dependent delay in the onset of resection provides time for recruitment of the HR proteins that normally curtail resection and direct the generated ssDNA towards homology search. The absence of Ku allows resection often to escape this control. Appropriate placement of INT markers should allow the extent of resection to be addressed in more detail in future work. We conclude with a note concerning the new visualization tool that enabled us to obtain these results. The ParB-INT visualization system allows direct kinetic measurements on single cells, avoiding interference with the process under study. These features should make the system widely applicable to the study of fine-scale chromosome positioning and dynamics in contexts beyond the repair process studied here, such as gene expression, replication and recombination.
When chromosomes break , cells must repair them to avoid becoming abnormal , cancerous or dead . The most accurate repair mechanism is based on homologous recombination ( HR ) , in which single strands generated next to the break seek an intact replica which is copied into the broken site . Changes in chromosome dynamics during the early steps that create the single strands have not been analysed owing to lack of tools allowing analysis of this process in individual living cells . We have developed a method for directly observing the resection process that prepares DNA double-strand breaks for HR . This allows us for the first time to identify just those cells where breaks are being repaired , and so to analyze the repair mechanism with a precision not attainable using current visualization systems . We have observed that the broken DNA is prepared for restoration much faster than previously thought , and that DNA movement first slows dramatically , prior to the more pronounced movement previously seen to accompany later stages of repair .