How Are Double Strand Breaks Repaired

Introduction

Among the factors that threaten genomic stability and integrity, Deoxyribonucleic acid double-strand breaks (DSBs) are peculiarly lethal. Induced by exogenic (ultraviolet [UV], ionizing radiation [IR], etc) or endogenic (alterations in Deoxyribonucleic acid metabolic reactions: replication; repair; etc) sources, DSBs must be repaired in all living organisms in guild for chromosome replication and transcription to proceed. DSBs are eliminated predominantly by nonhomologous end joining (NHEJ) and homologous recombination (Hr). During NHEJ, ii ends of a break are aligned and ligated, thus restoring DNA continuity without requiring an intact copy of the affected locus though, in upshot, causing mutations. Still, the most universally distributed DSB repair pathway is Hour, mediated past evolutionarily conserved recombinase proteins (RecA, RadA, and Rad51 [Dmc1] from bacteria, archaea, and eukaryotes, respectively), which repair a broken chromosome using an intact copy as a template in an error-free procedure.

In Escherichia coli, HR is the exclusive DSB repair pathway during which the RecA recombinase binds single-strand (ss)Dna derived from a break, thus creating a RecA nucleoprotein filament (the main recombination intermediate). The nucleoprotein filament subsequently performs a homology search and exchanges the broken DNA sequence with its intact homologue.¹ Since recombinases have lower affinity for ssDNA than their cognate ssDNA-binding proteins, SSB/RPA, recombinase polymerization on ssDNA is mediated past a recombination mediator class of proteins in a regulated mode.² These are the RecBCD and RecFOR proteins in bacteria, and BRCA2, PALB2, and Rad52 in eukaryotes.² The critical footstep in recombinase–nucleofilament assembly is the cosmos of a ssDNA fragment in cleaved DNA, onto which the recombinase tin demark. DSBs are thus processed past a combination of helicase/nuclease activities that lead to 3′-end overhangs, a substrate for recombinase bounden. This process involves nucleolytic deposition of a complementary five′-strand and is referred to as end resection.³ There are several pathways of Dna end resection in Due east. coli, and these have been traditionally used to define the 60 minutes pathways.¹

RecBCD pathway and its variants

In wild-blazon E. coli, a multifunctional heterotrimeric RecBCD enzyme repairs a bang-up majority of DSBs.¹ It binds blunt or nearly blunt double-strand (ds)DNA ends with high affinity and, with a combination of fast and processive helicase and nuclease activities, unwinds and degrades both strands of a DNA duplex (Figure 1A).⁴ RecB and RecD subunits are helicases, with RecB also having a unique nuclease and RecA-loading domain.^5–viii The destructive mode of RecBCD's action (which is useful for destroying foreign Dna) is converted to a productive mode by the enzyme's interaction with a regulatory octanucleotide Dna sequence Chi, whereupon the enzyme continues unwinding Deoxyribonucleic acid and degrading a five′-terminated strand, while leaving a iii′-mail service-Chi strand intact.^nine Moreover, Chi-modified RecBCD starts facilitating RecA loading onto such a created iii′-overhang.¹⁰ In this mode, the coupled helicase/nuclease/RecA-loading activities of RecBCD are sufficient for creating a RecA nucleoprotein filament in cleaved DNA.

Figure one DNA end resection pathways in Escherichia coli.
Notes: (A) In wild-type bacteria, a RecBCD enzyme binds a flush Dna stop and unwinds DNA duplex while simultaneously degrading both unwound strands. Afterward interaction with the Chi (χ) sequence in the DNA, the RecBCD is modified; its helicase activity is slowed downwards, and its nuclease activity is restricted to the 5′-terminated strand. Too, Chi-modified RecBCD starts facilitating RecA polymerization onto the three′-terminated strand, thus creating the recombinogenic RecA nucleofilament. The RecA nucleofilament is able to invade the homologous Dna sequence, during which a D-loop structure is formed. (B) In recD mutants, the RecBC enzyme unwinds Deoxyribonucleic acid duplex starting from a edgeless DNA finish. RecBC helicase lacks nuclease activity and constitutively loads RecA protein onto the unwound three′-terminated strand. A complementary five′-strand is degraded by ssExos, RecJ and ExoVII, which dethrone ssDNA with 5′–iii′ polarity. (C) In recB1080 mutant cells, the nuclease and RecA loading-deficient RecB¹⁰⁸⁰CD enzyme unwinds large tracts of DNA duplex. Unwound strands are bound past SSB proteins and degraded by ssExos, ExoI and RecJ, which trim three′- and 5′-strands, respectively. The unwound iii′-terminated strand may be involved in either RecA-dependent (a) or RecA-independent (illegitimate) (b) exchange. RecQ helicase and ExoI reduce the reactivity and longevity of the recombinogenic 3′-terminated tail, thus directing it toward homologous recombination and away from illegitimate recombination. RecA is polymerized onto the iii′-overhang with the help of RecFOR proteins. Hatched arrows denote the nonobligatory, sporadic reaction steps. (D) In cells lacking RecBCD, ExoI, and SbcCD enzyme functions, RecQ (or UvrD) acts as a helicase. The unwound strands are bound by the SSB protein and the 5′-terminated tail is trimmed by the RecJ exonuclease. RecFOR proteins facilitate an exchange of SSB for the RecA protein on the 3′-overhang, turning it into RecA nucleofilament. (E) recA mutants are homologous recombination scarce. Activities of several ssExos of iii′–5′ polarity – namely, ExoI, SbcCD, and ExoVII – are required for RecBCD binding to and degradation of the Dna duplex in a "reckless" manner, which preserves viability of the mutant.
Abbreviations: ExoVII, exonuclease VII; ExoI, exonuclease I; ssExos, unmarried-strand Dna-specific exonucleases; ssDNA, unmarried-strand Dna.

In recD nil mutants, DSBs are efficiently repaired by the RecBC enzyme even though it has no nuclease activity and does not collaborate with a Chi sequence.^one RecBC unwinds the Deoxyribonucleic acid duplex and loads RecA constitutively onto the 3′-tail (hence, behaving similarly to the Chi-modified RecBCD, except that it lacks v'–3′ exonuclease activity) (Figure 1B).^11,12 The RecBC'southward missing 5′–three′ exonuclease activeness is effectively compensated for by the action of RecJ and exonuclease (Exo)VII, cellular exonucleases that dethrone ssDNA (ssExos) from a 5′-end.¹³ RecJ is the dominant five′–3′ ssExo with ExoVII (which also possesses 3′–v′ exonuclease activity) providing backup activity.¹³ The importance of trimming the 5′-strand of a dsDNA cease is evident in a recD mutant deficient in RecJ and ExoVII, which is barely feasible and is recombination deficient, as well equally extremely sensitive to UV and gamma irradiation.^xiii,14

Another RecBCD-derived pathway is operative in recB1080 (and recB1067) mutants, which produce the RecB¹⁰⁸⁰CD (RecB¹⁰⁶⁷CD) enzyme. A unmarried amino acrid commutation in the agile heart of the RecB nuclease domain renders these enzymes nuclease and RecA-loading deficient, while leaving their DNA binding and helicase activities unaffected.^7,15 In these cells, RecB¹⁰⁸⁰CD provides rapid and processive helicase activity, while RecJ exonuclease and RecFOR proteins perform 5′–iii′ exonuclease and RecA-loading activities, respectively (Figure 1C).^16,17 The resulting HR and Deoxyribonucleic acid repair processes in the recB1080 mutant are less efficient, which results in its lower viability and increased sensitivity to DNA-damaging agents when compared to wild-blazon bacteria.^sixteen

The nuclease domain of the RecB subunit is connected to the helicase domain by a long linker,¹⁸ which contributes to the flexibility in its substrate specificity. The RecBCD nuclease domain degrades both of the unwound strands; all the same, it switches to degradation of but the 5′-terminated strand upon the enzyme's interaction with a Chi site in the DNA. Also, the RecBC enzyme assumes a type of conformation that renders information technology nuclease scarce, fifty-fifty though information technology possesses an intact nuclease domain.

RecF pathway

In contrast to RecBCD'southward action on DSBs, RecFOR proteins in wild-type E. coli mediate the polymerization of RecA on internal ssDNA regions or gaps, which originate mainly from replication defects.¹⁹ E. coli mutants lacking the RecBCD enzyme are poorly viable and are highly sensitive to genotoxic agents that induce DSBs, unless ssExos of proven (ExoI) and apparent (SbcCD) 3′–five′ polarity are inactive.¹ In this genetic background, a RecQ helicase and, to a lesser extent, a UvrD helicase unwind pause ends (a role for a HelD helicase has been ruled out recently),^twenty thus enabling RecJ-catalyzed degradation of the unwound 5′-overhang, while its 3′-complement is bound past RecA with the help of RecFOR proteins (Figure 1D).¹ Hence, inactivation of ExoI and SbcCD ssExos allows for the combined activity of several helicases/nucleases/recombination mediator proteins to finer supercede the RecBCD function in DSB repair. This demonstrates the robustness of the E. coli DSB repair capability, which is not surprising because the importance of DSB removal for jail cell survival.

A RecA nucleoprotein filament formed by any of the same pathways searches for an intact homologous sequence and invades it, thus creating a deportation loop (also chosen a D-loop) structure.¹ Ensuing strand exchange reactions and replication initiated by PriA turn this three-strand recombination intermediate into a replication fork,²¹ when a unmarried dsDNA cease (eg, a collapsed replication fork) is repaired. Alternatively, when two dsDNA ends of a DSB are processed, they requite rising to a pair of iv-strand structures designated as Holliday junctions. Finally, the HR procedure is completed by resolving the Holliday junctions through the deportment of the RuvABC resolvase,²² thus producing mature recombination products.

Real-time monitoring of DSB repair in E. coli

By using an I-SceI endonuclease system for chromosomal DSB induction in combination with fluorescently marked proteins, a recent report²³ has characterized the progression of DSB repair in live Due east. coli cells. This revealing study has shown that a DSB in a site-specific chromosome locus is repaired using afar sister homology as a template. In wild-type cells, RecBCD processing of both ends of a DSB precedes RecA polymerization, which nucleates into big structures that the authors term bundles.²³ Once formed, two RecA bundles apace relocate toward uncut homologous loci, with which they pair.²³ Throughout all of these reactions, the 2 ends of a DSB are kept in close proximity, suggesting that both of them participate in DSB repair, equally seen in Hour-dependent DSB repair in eukaryotes.²⁴ The written report revealed that RecN, a relatively poorly characterized DSB repair protein, is recruited to a DSB early, which is in accord with an earlier study showing that a RecA nucleofilament mobilizes RecN.²⁵ In addition, every bit expected, PriA and RecG were shown to be recruited to a DSB later, thus confirming that their functions in replication restart and processing of Holliday junctions, respectively, are required for DSB repair.²³ Lastly, the kinetics of DSB repair in E. coli was determined:²³ RecBCD degradation of the ends of a DSB lasts for ~60 minutes, during which time RecA bundles course starting at ~5 minutes postcut, and reaching a maximum at ~fifteen minutes postcut. Bundle relocation lasts for ~47 minutes, followed by sis loci pairing, which takes ~5 minutes; then, in another ~17 minutes, the bundles are disassembled. Sis pairing persists for ~l minutes before their segregation. Overall, it takes ~150 minutes to repair a DSB in wild-type East. coli.²³ Even so, as the cells in this study were grown at 30°C and microscopy was performed at 24°C, it is likely that DSB repair takes less than 150 minutes at the physiological temperature of 37°C.

A slight change in RecA protein strongly increases E. coli resistance to high doses of ionizing radiations

When Eastward. coli was repeatedly subjected to 3,000 Gy of IR in directed evolution experiments, after twenty such cycles, the survivors became 3–4 orders of magnitude more radioresistant than the initial population.²⁶ recA is one of the three genes involved in DNA metabolism whose mutation leads to a majority of the extreme resistance.²⁷ Proteins produced past such mutated recA alleles, RecA^D276N and RecA^D276A, increase IR resistance of the starter strain by approximately tenfold.²⁷ Biochemical characterization of the 2 altered proteins revealed that they are less sensitive to inhibition by adenosine diphosphate. They also nucleate a RecA nucleoprotein filament more than rapidly, merely extend information technology more than slowly than wild-type RecA.²⁷ As a outcome, a college number of shorter RecA nucleoprotein filaments can be formed in a cell. This is certainly beneficial for the repair of a large number of DSBs caused by iii,000 Gy of IR. This example shows that East. coli has the capacity to repair many more than DSBs than information technology ordinarily does, with but slight alterations (a unmarried amino acid change in this case) in the existing repair pathways. However, every bit E. coli hardly always encounters such high levels of IR, wild-type RecA is optimized for repairing a low number of simultaneous DSBs in a cell, for which longer RecA nucleofilaments might be favorable because they enable more rapid homologous Deoxyribonucleic acid pairing.²⁸ In this respect, ane would expect that the viability and Dna repair efficiency of the recA276 mutants exposed to depression doses of genotoxic agents is worse than those of their parental wild-type cells, and also that the recA276 mutants would be outgrown in competition assays with wild-type bacteria when IR is not applied.

three′-overhang metabolism matters in Eastward. coli DSB repair

While the significance of nucleolytic degradation of 5′-terminated strands emanating from a DSB is well appreciated, DNA terminate resection is divers past it; contempo studies, which volition be discussed, have pointed out the importance of complementary 3′-end strand metabolism in HR. Several aspects of 3′-end metabolism depict Deoxyribonucleic acid end resection as a rather complicated process, which cannot be confined to simply five′-strand degradation.

The balance of helicase and exonuclease activities in 60 minutes

In RecBCD-deficient cells, as described in the RecF pathway section, the inactivation of 3′–5′ ssExos, ExoI and SbcCD, enables DSB repair, with RecQ and RecJ providing 3′–5′ helicase and v′–3′ exonuclease action, respectively. RecQ is a weak helicase (unwinds ~2 base pairs per 2d)²⁹ and it certainly cannot compete with the strong, processive 3′–5′ ssExos ExoI (trims ~275 nucleotides per second)^thirty and SbcCD; hence, no HR occurs in RecBCD⁻ ExoI⁺ SbcCD⁺ cells. However, Buljubašić et al²⁰ accept recently shown that in RecBCD⁻ ExoI⁻ SbcCD⁻ mutant, the additional inactivation of ExoVII, a processive 3′–five′ ssExo,³¹ relieves the requirement for the RecQ helicase in Hour. On the other manus, if a distributive 3′–5′ ssExo ExoX³² is inactivated instead of ExoVII, the RecQ requirement is not alleviated.²⁰ Hence, in recBCD mutants, a requirement for helicase activeness in DSB repair depends on the amount of iii′–5′ exonuclease action – ie, the weaker the 3′–5′ exonuclease activity is, the less the requirement for the helicase action. An in vitro RecF pathway reconstruction written report³³ has revealed like effects; namely, in experiments defective iii′–5′ ssExos, RecQ is loosely required and RecJ solitary can produce long enough ssDNA from a dsDNA end to enable a RecA-catalyzed strand invasion, admitting at lower efficiency than in a RecQ-assisted reaction. This means that in the absence of 3′–five′ exonuclease activity, helicase activity becomes dispensable for the RecF pathway of Hour due to RecJ's ability to dethrone dsDNA.³³ Yet, since HR and Deoxyribonucleic acid repair are very efficient in RecBCD⁻ ExoI⁻ SbcCD⁻ ExoVII⁻ RecQ⁻ UvrD⁻ mutants,^twenty wherein a 3′-tail is tending to the action of 3′–five′ ssExos (eg, ExoX, PNPase,³⁴ RNase T,³⁵ etc), it is quite likely that another helicase(s) process(es) dsDNA ends in these cells.

The aforementioned is a situation where the combination of weak helicase and stiff iii′–5′ exonuclease activity prevents HR. Some other report³⁶ has recently described the contrary situation, in which a helicase is so strong that it outperforms the 3′–5′ ssExos; this situation is also deleterious for a prison cell. Namely, in the recB1080 mutant, a highly processive (estimated at ≥50 kbp/binding event) and fast (1–ii kbp/s) RecB¹⁰⁸⁰CD helicase produces tens of kb long overhangs.³⁶ Jail cell viability, DNA repair, and HR of the mutant are impaired when compared to the wild-type strain, with further deterioration observed upon inactivation of 3′–five′ ssExos ExoI,³⁶ ExoVII, SbcCD, and ExoX (Đermić, unpublished data). A deleterious effect of increased processivity of Dna end-processing, resulting in longer iii′-overhangs, was noted in eukaryotic cells equally well, which showed increased genomic instability and higher sensitivity to Dna harm.^37–39

Together, these situations bear witness that the coordination of helicase and iii′–5′ exonuclease activity is essential for efficient DSB processing in E. coli. An imbalance in that coordination may issue in either an inability to create a 3′-overhang (when the 3′–5′ exonuclease activity is dominant), or in the production of a very long iii′-tail (when helicase activity dominates), which is reactive and tends to engage in aberrant, nonproductive reactions (which volition be discussed). This argument provides the rationale for the coupling of robust nuclease activity to strong helicase activity in the RecBCD enzyme. From that perspective, RecBCD nuclease action'southward raison d'être is primarily DSB repair rather than degradation of foreign DNA. Many phages have adult protection mechanisms against RecBCD, while leaner take developed RecBCD-independent adaptive amnesty against invasive exogenic genetic elements.⁴⁰ During DSB repair, RecBCD nuclease action prevents long 3′-terminated overhangs from appearing in a jail cell, while continuously removing 5′-strands. Indeed, in the RecBCD auto, the fast and processive helicase action is coupled to powerful exonuclease activity on both unwound strands until interaction with a Chi site marks the Deoxyribonucleic acid duplex equally a host molecule that should be repaired instead of destroyed. At that betoken the Chi-modified enzyme slows down and stops degradation of the 3′-strand while continuing deposition of the 5′-strand.^1,4 Although the Chi-modified RecBCD is slowed down, the recombination reactions it catalyzes are insensitive to ExoI, the strongest iii′–5′ ssExo,³⁶ thus suggesting that the enzyme has optimally coordinated helicase and nuclease activities. In improver, the polymerized RecA protein may protect a post-Chi 3′-overhang from the activeness of ExoI.^xi

Remarkably, there is an analogous instance of balancing helicase and nuclease activities in the eukaryotic stop-resection protein, Dna2, which possesses both 5′–3′ helicase and 5′–iii′ nuclease activity.⁴¹ During end resection, the nuclease activeness of this RecB related protein is coupled to the helicase activity of the RecQ-like helicases, Sgs1 and BLM, while its ain helicase activeness is non required.^42,43 Saccharomyces cerevisiae Dna2 deficient in nuclease activity is a vigorous helicase, suggesting that its nuclease activity suppresses its helicase activity.⁴⁴ A structural switch in Dna2 that regulates the residuum betwixt its helicase and nuclease activities remains to be determined.

Coordination of helicase and RecA-loading action in RecBCD determines the efficiency and fidelity of DSB repair

In add-on to beingness a kind of "runaway" helicase devoid of nuclease activity, RecB¹⁰⁸⁰CD is also deficient in the facilitation of RecA loading.^fifteen That makes information technology more than akin to the DNA terminate resection machines in eukaryotes, which also have uncoupled helicase/nuclease and recombinase-loading activities (different RecBCD).³⁶ The recB1080 mutant is thus a convenient E. coli model for studying eukaryotic Deoxyribonucleic acid end resection mechanisms.³⁶ In this respect, information technology is interesting that inactivation of the RecQ helicase impairs prison cell viability, Dna repair, and Hr of the recB1080 mutant, thus resembling sickly phenotypes caused in eukaryotes by deficiencies in RecQ'due south eukaryotic analogs.³⁶ Also, the deleterious effect of ExoI inactivation in the recB1080 mutant, equally described earlier, is exacerbated by inactivation of the RecQ helicase.³⁶ A recB1080 mutant lacking ExoI and RecQ is poorly viable, 60 minutes and DNA repair scarce and, notably, displays highly increased illegitimate recombination.³⁶ The deleterious effects of ExoI and RecQ inactivation are non observed in wild-type and recD genetic backgrounds,³⁶ which brandish the helicase/nuclease/RecA-loading and helicase/RecA-loading activities of RecBC(D), respectively. Therefore, information technology may be inferred that the combined helicase and RecA-loading activity of the RecBC(D) enzyme funnels three′-overhangs into productive 60 minutes reactions and away from aberrant, nonproductive reactions, thus preventing illegitimate recombination.³⁶

In improver to its role in the initiation of the RecF pathway of Hr, RecQ also binds to recombination intermediates, such as Holliday junctions and joint molecules in Kappa and D-loop formation,⁴⁵ which it disrupts, especially those arising from aberrant, nonhomologous joint exchanges.^46,47 RecQ and its widespread orthologs are structurally and functionally highly conserved, and members of this poly peptide family participate in both Hr initiation and disruption/regulation, thus interim as key genome caretakers from leaner to humans.^47,48

In the recB1080 ExoI⁻ RecQ⁻ mutant, long iii′-overhangs produced by the fast and processive RecB¹⁰⁸⁰CD helicase have increased longevity due to ExoI inactivation, and they are too more reactive considering of RecQ deficiency (Figure 1C), which is very deleterious for the cell.³⁶ These effects show that the metabolism of the 3′-terminated strands is seemingly equally every bit of import every bit deposition of the 5′-terminated strands for efficient and precise DSB repair in E. coli and, hence, for its genome stability.

Interestingly, RecBCD's Bacillus subtilis analogue, AddAB, does non facilitate RecA polymerization on a 3′-tail, which is catalyzed past RecOR mediators instead.⁴⁹ The AddAB class of helicase/nuclease proteins is present in considerably more bacterial species than the RecBCD class.^fifty This means that intimate coupling of helicase and recombinase-polymerizing activities occurs in the DSB cease resection apparatus of only a minority of bacteria, while eukaryotic organisms, archaea, and the majority of bacteria take looser coordination between the ii activities and, consequently, they take evolved dissimilar mechanisms for assuring precise and efficient DSB repair. Therefore, it is likely that due to uncoupling of the helicase and RecA-loading activity of AddAB (analogous to RecB¹⁰⁸⁰CD), a function of three′–5′ ssExos and RecQ helicase (and RecOR proteins) is more prominent in preserving genome stability in bacterial species that harbor AddAB than in those harboring the RecBCD enzyme.

3′–5′ ssExos control DSB processing in a recA mutant

Although a recA null mutant is HR deficient, the RecBCD enzyme nevertheless helps maintain its viability.^iv This beneficial office is due to RecBCD'due south exonuclease action, which is in fact unregulated, "reckless", and may pb to the deposition of a complete chromosome.⁵¹ It has recently been shown that RecBCD-catalyzed Dna degradation in the recA mutant is dependent on three′–5′ ssExos ExoI, SbcCD, and ExoVII.^52,53 Genetic prove suggests that these ssExos are required for loading and/or reloading of the main DSB processing car RecBCD to dsDNA ends in recA cells (Figure 1E).^52,53 And since RecBCD loading to DNA is not dependent on ExoI, SbcCD, and ExoVII in wild-type cells,^52,53 one can conclude that dsDNA ends in recA cells, unlike those in wild-type bacteria, contain long 3′-overhangs that prevent RecBCD loading (Figure 1E). Two sources for such ends occurring in recA cells are proposed: a regressed replication fork would requite rise to a dsDNA terminate with a long iii′-tail in the absence of the RecA poly peptide; and/or a mail-Chi 3′ tail created past RecBCD in recA leaner is not covered by the RecA protein and is therefore not immobilized in a HR intermediate, nor is it protected from degradation by ExoI, and possibly other 3′–5′ ssExos.^52,53

The same role of the SbcCD exonuclease in enabling loading of the chief DSB processing motorcar to DSBs is especially interesting, equally it mimics the part of SbcC and SbcD eukaryotic orthologs, Rad50 and Mre11, respectively, in bringing about recruitment of the chief DSB processing proteins that perform long-range DSB finish resection.^three This, in plow, directs DSB repair toward HR and, to a lesser extent, microhomology-mediated end joining repair pathways, and away from NHEJ.⁵⁴ Nonetheless, the difference between bacterial SbcCD and its eukaryotic equivalents is that the onetime participates in trimming of 3′-tails (every bit suggested by aplenty genetic bear witness),^13,20,51,55 whereas the latter performs initial brusque-range DSB end resection by degrading five′-strands, fifty-fifty though it as well possesses exonuclease activity of the 3′–5′ polarity.⁵⁶

Acknowledgments

The author is supported by Croatian Science Foundation, project 2978. I am grateful to Nikola Paić and Mary Sopta for their assistance with manuscript preparation.

Disclosure

The writer reports no conflicts of interest in this piece of work.

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