DNA end resection: nucleases team up with the right partners to initiate homologous recombination

The repair of DNA double-­‐strand breaks (DSBs) by homologous recombi-­‐ nation commences by nucleolytic degra-­‐ dation of the 5'-­‐terminated strand of the DNA break. This leads to the formation of 3'-­‐tailed DNA, which serves as a sub-­‐ strate for the strand exchange protein Rad51. The nucleoprotein filament then invades homologous DNA to drive tem-­‐ plate-­‐directed repair. In this review, I discuss mainly the mechanisms of DNA end resection in Saccharomyces cere-­‐ visiae , which includes short-­‐range resec-­‐ tion by Mre11-­‐Rad50-­‐Xrs2 and Sae2, as well as processive long-­‐range resection by Sgs1-­‐Dna2 or Exo1 pathways. Resec-­‐ tion mechanisms are highly conserved between yeast and humans, and analo-­‐ gous machineries are found in prokary-­‐ otes as well.

DSBs commits their repair to HR as it pre-vents ligation by the potentially more muta-genic non--homologous end--joining (NHEJ) pathway (2--4). Resected DNA is first coated by the ssDNA binding protein Replication Protein A (RPA). In most cases, RPA is sub-sequently replaced with the strand ex-change protein Rad51, forming a nucleopro-tein filament capable to invade homologous DNA. Repair can then proceed via either of two main recombination pathways, synthe-sis--dependent strand annealing (SDSA) or the canonical pathway that involves the formation of a double Holliday junction (DSBR) (Fig. 1). Single--strand annealing (SSA) is instead a Rad51--independent pathway that requires extensive resection of DNA between two repetitive sequences ( Fig.  1).

DNA end resection: when and what to resect
DSBs can form accidentally in any phase of the cell cycle upon exposure to ionizing radiation, chemicals or as a result of abor-tive processing of nucleic acids. Most DSBs however occur in S--phase when a DNA rep-lication fork runs into a nick. Furthermore, DSBs are sometimes introduced "intention-ally" such in the prophase of the first meiot-ic division or during anti--cancer therapy regimens based on DNA damaging agents (5). Depending on the cellular context, cells must first "decide" whether or not to resect the breaks (3,4,6,7). DNA end resection commits the repair to HR and prevents NHEJ; therefore, it would be detrimental to resect DSBs in the G1 phase of the cell cycle when no sister chromatid DNA is available as a template for repair. Cells have thus de-veloped regulatory control mechanisms that activate resection only during the S or G2 phases of the cell cycle, which will be intro-duced below (4,6--8).
Another critical parameter is the polari-ty of resection. It has been observed in vivo that the 5'--terminated strand of the dsDNA break is specifically resected (9,10). Alt-hough limited processing of the 3'-terminated strand has been observed as well (11), the preferential degradation of the 5'--terminated strand results in the for-mation of 3'--tailed DNA. This becomes a substrate for Rad51 and upon strand inva-sion the 3' end primes DNA synthesis, which is required for the downstream steps in the HR pathway (1). How the various DNA end processing machineries ensure the 5'→3' polarity of resection will be discussed.

DNA end resection: lessons from prokaryotes
In Escherichia coli, DNA end resection is carried out either by the RecBCD-- or RecQJ-dependent pathways (12). RecBCD is a vig-orous nuclease--helicase complex with a strong affinity towards DNA ends. RecB has a helicase activity that unwinds DNA in a 3'→5' direction, which functions synergisti-cally with the RecD helicase subunit. RecD translocates on the opposite strand than RecB with a 5'→3' polarity resulting in a net translocation in the same direction away from the DNA end (13--15). The RecB and RecD motors do not run at the same speed. The unique bidirectional translocation mechanism gives rise to a ssDNA loop that accumulates in front of the slower RecB subunit, and which is detectable by electron microscopy (15). Before encountering the regulatory Chi (crossover hotspot instiga-tor) sequence within genomic DNA, the RecB subunit degrades both 5' and 3'-terminated DNA strands. Upon encounter-ing Chi, the complex pauses and continues translocating at a reduced speed dependent on RecB, which becomes the lead motor (13). Importantly, the nucleolytic degrada-tion of the 5'--terminated DNA is upregulat-ed, while the degradation of the 3'-terminated strand is attenuated, which de-termines the polarity of DNA end resection (16). DNA without a Chi sequence is fully degraded by RecBCD, which contributes to prokaryotic defense mechanisms against invading DNA. Furthermore, RecBCD direct-ly loads the strand exchange protein RecA on the arising 3'--tailed DNA, which facili-tates recombination (17).
The RecQJ enzymes initiate a second ma-jor recombination pathway in E. coli, which also requires the RecFOR factors (12). RecQ, a founding member of the RecQ helicase family, unwinds dsDNA with a 3'→5' polari-ty, which generates ssDNA for the RecJ nu-clease that degrades DNA 5'→3' in a manner stimulated by the ssDNA binding protein SSB (12,18). Therefore, unlike RecBCD, the activity of the RecQJ complex directly pro-duces 3'--tailed DNA and the resection polar-ity is not regulated by the Chi sequence. As RecBCD, the RecFOR complex also loads Re--cA on the SSB--coated ssDNA at junctions of single and double--stranded DNA (19).
While the RecFOR pathway is conserved across prokaryotes, the RecBCD complex is only present in some bacteria that include gram--negative E. coli (20). In gram--positive Bacillus subtilis, RecBCD is replaced by the AddAB complex (20). AddAB has a single motor within the AddA subunit that un-winds DNA with a 3'→5' polarity, which is stimulated by the AddB subunit (21). While no Chi sequence has been detected in eu-karyotes, variations of similar helicase-nuclease complexes that resect DSBs are conserved in evolution.
3. Two--step resection model: the rela-tionship between short and long--range resection pathways DNA end resection in eukaryotes is a two--step process in most cases (9,22,23). It is initiated by a nucleolytic processing step that is slow and limited to the vicinity of DNA ends (9,23). In S. cerevisiae, this first step is dependent on the Mre11--Rad50--Xrs2 (MRX) complex. MRX has an affinity for DNA ends, and was shown to be one of the first proteins recruited to DSBs (24,25). It has both catalytic and structural roles in DNA end processing. The intrinsic nuclease activ-ity of Mre11 is capable to degrade 5'-terminated DNA in the vicinity of the DNA end. The structural role of MRX involves re-cruitment of components belonging to the second long--range processing step (9,23,26--29). In yeast, these include two separate pathways dependent on either the Sgs1--Dna2 helicase--nuclease or the Exo1 nucle-ase (Fig. 2).
DSBs arise in multiple ways and thus are very diverse in structure (5). Some are chemically "clean" and may either be blunt-ended or have short 5' or 3' ssDNA over-hangs. These stretches of ssDNA may form secondary structures that impede resection. Many DSBs are chemically "dirty", including those induced by ionizing radiation, which in addition to DNA breakage gives rise to oxidative DNA damage. Furthermore, DSBs can arise upon abortive topoisomerase reac-tions that may occur either spontaneously or upon drug treatment. E.g. the anti--cancer drug etoposide inhibits Topo II, which re-mains trapped at the 5'--terminated strand of the DSB (30). Finally, DSBs in meiosis are introduced by the Topo II--like enzyme Spo11, which also remains covalently at-tached to the 5' end of the broken DNA (31--33). The presence of secondary structures, chemical modifications or proteins at the DNA end represents a specific challenge to the resection machinery. It has been demonstrated that the short--range resec-tion pathway, and specifically the nuclease of Mre11, is required for the processing of these non--canonical DNA ends (26,34,35). The Mre11 nuclease activity is instead largely dispensable for the resection of en-donuclease--induced "clean" DSBs (36) (Fig.  2). Similarly, the structural role of MRX is not essential, as Exo1 and Sgs1--Dna2 can initiate resection of clean DNA ends in MRX-independent manner however less efficient-ly (9,27--29,37).
Long--range resection pathways were ini-tially identified using physical assays that measure the kinetics of ssDNA formation at various distances from an experimentally induced dsDNA break (9,23). In order to improve detection, these assays were per-formed in a rad51Δ background that does not allow the repair of the DSB. In addition, genetic assays based on SSA were utilized, in which long tracts of DNA must be resect-ed to reveal a repeated sequence to allow repair (9,23). Together, these assays showed that Sgs1--Dna2 or Exo1 pathways are capable to resect very long stretches of DNA of more than 50,000 nts in length (9). Subsequent work revealed that these assays largely overestimated the length of DNA that is resected in vivo under normal condi-tions when repair is possible. In mitotic cells, it has been determined that ~2,000--4,000 nts are resected in allelic recombina-tion and ~3,000--6,000 nts in ectopic re-combination (38). In meiotic cells, where the long--range resection is largely depend-ent on Exo1, the resection tracks are even shorter (~800 nts) (39). In the sgs1Δ exo1Δ double mutant that is deficient in long--range resection, the degradation tracks are re-duced to ~100--300 nts in mitotic cells and 270 nts in meiotic cells (38,39). Intriguing-ly, the limited MRX and Sae2 dependent re-section is sufficient for efficient joint mole-cule formation in meiosis, and results in on-ly a moderate recombination defect in vege-tative cells (30--50% reduction) (9,38). Therefore, long--range resection is largely dispensable for recombination in meiosis, and not strictly required for repair in vege-tative cells, although it may be necessary for proper DNA damage checkpoint and maintenance. In gene targeting, elimination of the long--range resection pathways in-creased efficiency up to 600--fold (38). This demonstrated that Sgs1--Dna2 or Exo1 over-resected the transformed DNA. The short-range processing by MRX--Sae2 complex was sufficient for homology search and repair (38).

Short--range DNA end processing by MRX and Sae2: mechanism and regula-tion
The MRX complex likely functions as a dimer (40,41). It has a DNA binding activity with a preference towards DNA ends (24,42). The Rad50 subunit is an ATPase that controls conformation changes within the complex upon DNA binding, which regu-lates its functions in DNA end tethering, re-section and DNA damage signaling (43--45). In vitro, Mre11 is a manganese--dependent exonuclease that is moderately stimulated by Xrs2 (24). Mre11 also has a much weaker endonuclease activity on diverse secondary structures that is moderately promoted by Rad50 in the presence of ATP (46). Howev-er, the polarity of the Mre11 exonuclease (3'→5') was in disagreement with the polar-ity of resection observed in vivo (5'→3') as by guest on March 24, 2020 http://www.jbc.org/ Downloaded from well as with the DSB repair model that pos-tulates that 3'--tailed ssDNA tails must be generated (46--48). To this point, it was shown that P. furiosus Mre11--Rad50 has a weak magnesium--dependent endonuclease activity on the 5'--terminated strand near a DNA end (49). Later, it was demonstrated with recombinant S. cerevisiae proteins that Sae2 strongly promotes the endonuclease of Mre11 within the MRX complex (50). Simi-larly as in the Hopkins and Paull study (49), the endonuclease activity was magnesium-dependent and showed a preference to-wards 5'--terminated DNA. The preferential cleavage of the 5'--terminated DNA ~15--25 nucleotides away from the end suggested that the Mre11 nuclease initiates DNA re-section via its endonuclease, rather than exonuclease activity. Furthermore, the en-donucleolytic 5' end clipping was strongly promoted by protein blocks at the DNA end, demonstrating a possible mechanism of processing non--canonical DNA ends that are refractory to exonucleases (50). Under physiological conditions, when magnesium concentrations strongly exceed those of manganese and when DNA ends are pro-tected by a number of factors, the Mre11 exonuclease activity might be attenuated and MRX might preferentially function as a Sae2--promoted endonuclease (50).
The biochemical reconstitution experi-ments validated models that have been for a long time inferred from genetic studies. Specifically in meiosis, the Spo11 protein was found in complexes with oligonucleo-tide DNA molecules of ~12 and ~21--37 nu-cleotides in length (31,51). These DNA fragments were attached to Spo11 via their 5' end and had a free 3' DNA end, which suggested that the processing of meiotic DSBs is initiated by an endonucleolytic cut. The MRX complex was proposed as being the best candidate for the enigmatic nucle-ase. Subsequent studies revealed that end processing, at least in some cases, is initiat-ed by a cut at a position more distant from the DNA end, up to ~100--300 nucleotides away (52). This collectively provided sup-port for a bidirectional resection model, which posits that upon the initial endonu-clease cleavage, the Mre11 exonuclease pro-ceeds back towards the DNA end via its 3'→5' exonuclease activity (Fig. 2). At the same time, the endonuclease cut can create an entry point for the long--range resection enzymes. However, on the mechanistic level, it remains to be determined how the endo-nucleolytic cleavage by Mre11 is directed to the more distant sites away from the DNA break.
Genetic experiments also revealed that the Sae2 protein functionally integrates with the MRX complex (32). The pheno-types of sae2Δ cells resemble those of mre11 nuclease--deficient mutants in many genetic assays. In meiosis, sae2Δ strains are com-pletely deficient in the processing of Spo11-bound DNA breaks; furthermore, sae2Δ also affects Mre11 nuclease function in mitotic cells (32,53--56). This led to the notion that Sae2 might activate the nuclease of Mre11, as later directly demonstrated by reconsti-tution experiments (50). In contrast, cells lacking SAE2 are more sensitive than mre11 nuclease--dead mutants to DNA damaging agents (26). Thus, in addition to stimulating the Mre11 endonuclease, Sae2 has other, Mre11--nuclease independent roles. This may include its proposed function to re-move MRX from DNA ends upon end pro-cessing to facilitate downstream repair, at-tenuate checkpoint signaling, counteract the NHEJ factor Ku and promote resection by Exo1 (26,29,57--59). Sae2 itself was also shown to possess a nuclease activity specific to secondary structures in DNA (60), alt-hough an enzymatic activity was not detect-ed by other laboratories (27,50). Human and S. pombe Sae2 homologues (CtIP and Ctp1, respectively) were found to tetramer-ize, which was shown to be important for their function in vivo (61,62). Similarly, mu-tations that prevent oligomerization of Sae2 in vivo resulted in null phenotypes in several genetic assays (53). Intriguingly the nucle-ase of Sae2 has been suggested to be specific to its monomeric form (63). Taking togeth-er, the role of Sae2 in DNA metabolism is still only partially defined.
The Sae2 function in regulating the nu-clease of Mre11 makes it an ideal target for control by posttranslational modifications (8). Indeed, Sae2 is phosphorylated in S/G2 phases of the cell cycle by the cyclin-dependent protein kinase (CDK) Cdc28 (4,6). The key CDK target site is likely S267, which must undergo phosphorylation to allow resection both in vivo and in vitro (6,50). The phosphomimicking mutant Sae2 S267E partially rescues resection defect in the absence of CDK activity, while the non-phosphorylatable S267A mutant phenotype is comparable to that of sae2Δ cells (6). Therefore, the CDK--dependent regulation of Sae2 activity represents one of the key con-trol mechanisms ensuring that resection only takes place in the S/G2 phase of the cell cycle when a homologous template is avail-able for repair. In addition to CDK, Sae2 is also regulated by the Mec1 and Tel1 kinases in response to DNA damage (63--65). Phos-phorylation of Sae2 was shown to affect its oligomeric state (63). Furthermore, muta-tions of Mec1/Tel1 target sites to non-phosphorylatable residues in Sae2 result in DNA damage sensitivity, showing that also phosphorylation under the control of DNA damage checkpoint is important for the function of Sae2 in vivo (63--65). As Sae2 has additional roles on top of controlling Mre11 (see above), it remains to be determined whether the Mec1/Tel1--dependent phos-phorylation affects DSB resection or other functions of Sae2.
In higher eukaryotes, the homologue of MRX is the MRN complex, which consists of MRE11, RAD50 and NBS1 subunits (66,67). Similarly as in yeast, recombinant MRN has a manganese--dependent 3'→5' exonuclease and a weaker endonuclease activity (47,48,68). The human counterpart of Sae2 is CtIP, though the sequence homology is restricted to its C--terminal part as CtIP is a much larger protein than Sae2 (69). Exper-iments based on small molecule inhibitors that target specifically the endonuclease or the exonuclease of human MRE11 revealed that the endonuclease precedes the exonu-clease in resection (2). Thus, the bidirec-tional resection is likely conserved in evolu-tion and not limited to meiosis. However, whether and how CtIP regulates the MRE11 endonuclease has not been directly estab-lished yet. In contrast to yeast however, the activity of MRN and CtIP in DNA end resec-tion cannot be bypassed, as DNA end resec-tion is generally dependent on the presence of CtIP and the nuclease activity of MRE11 (69).

Long--range DNA end processing by Sgs1--Dna2 or Exo1
While the involvement of MRX in the processing of DNA ends has been known for a long time (70), the pathways responsible for the long--range resection were identified much later. This is most likely due to that fact that long--range resection can be carried out by either of two non--overlapping path-ways, dependent on the enzymatic activities of Sgs1--Dna2 or Exo1 (9,22,23). Inactivation of a single pathway results in only a minor resection defect, because the other pathway can effectively compensate. Major resection defects were only revealed when both pathways were inactivated simultaneously, e.g. in sgs1Δ exo1Δ double mutants (9,22,23).

Sgs1--Dna2 resection pathway
Both Sgs1 and Dna2 have separate func-tions unrelated to DNA end resection. Sgs1 is a vigorous DNA helicase belonging to the RecQ family (71,72), which functions to-gether with Top3 and Rmi1 to dissolve dou-ble Holliday junctions into non--crossover products, thereby preventing sister-chromatid exchanges and chromosome in-stability (73,74). Dna2 is a bifunctional hel-icase--nuclease responsible for removing DNA flaps arising by strand displacement synthesis by DNA polymerase δ during lag-ging strand DNA synthesis (75). The Oka-zaki fragment processing function of Dna2 is essential, although the viability of dna2Δ mutants can be rescued by multiple mecha-nisms (76). Prior to the seminal work by Ira and colleagues (9), Sgs1 and Dna2 had not been implicated to function together.
The mechanism of DNA end resection by the Sgs1--Dna2 pathway was revealed by a combination of genetic and biochemical ex-periments. The helicase of Sgs1 unwinds dsDNA with a 3'→5' polarity, which pro-vides a substrate for the ssDNA--specific Dna2 nuclease (9,27,28). Dna2 must load on a free ssDNA end but then degrades DNA endonucleolytically resulting in degradation products of ~5--10 nucleotides in length (77). Dna2 was shown to possess both 3'→5' and 5'→3' nuclease activities (78), so its in-volvement in DNA end resection was initial-ly puzzling. The issue was resolved later when it was demonstrated that RPA inhibits by guest on March 24, 2020 http://www.jbc.org/ Downloaded from the degradation of 3'--terminated ssDNA, while it stimulates the degradation of the 5'-terminated strand (27,28). Therefore, RPA is a crucial factor that enforces the correct polarity of DNA end resection by the Sgs1--Dna2 pathway, leading to the production of 3'--tailed DNA (Fig. 3a).
Dna2 also possesses a DNA helicase ac-tivity with a 5'→3' polarity. Unlike the nu-clease of Dna2 that is essential for cell via-bility, helicase--deficient dna2 mutants are viable under certain growth conditions (76). The physiological role of the Dna2 helicase is not yet clear. The DNA unwinding activity of Dna2 is vigorous, comparable to the hel-icase capacity of Sgs1, yet it is cryptic and only becomes apparent upon inactivation of the Dna2 nuclease (79). It is tempting to think that the helicase of Dna2 functions in concert with that of Sgs1 (28). Both Sgs1 and Dna2 were shown to directly interact, which led to the model where Sgs1 translo-cates along one DNA strand in a 3'→5' direc-tion and unwinds DNA, whereas Dna2 trans-locates with a 5'→3' polarity on the second DNA strand unwound by Sgs1, yet in the same general direction as Sgs1 (28). This mode of translocation and DNA degradation would be reminiscent of the resection com-plexes from bacteria such as RecBCD (14); though, it has not been substantiated bio-chemically. Specifically, in contrast to a bidi-rectional manner of DNA translocation by Sgs1--Dna2, the helicase activity of Dna2 was implied to be dispensable for DNA end re-section (9). Similarly to the nuclease domain of B. subtilis AddA, also Dna2 contains a 4Fe--4S iron--sulfur cluster that appears to have a structural role, which further highlights the parallels between prokaryotic and eukary-otic resection complexes (21). More exper-iments are clearly needed to determine whether and how the helicase of Dna2 with-in the Sgs1--Dna2 heterodimeric complex promotes resection.
Several factors have been identified that stimulate DNA end resection by Sgs1--Dna2, which includes the MRX complex and the Top3--Rmi1 heterodimer (9,27,28). As dis-cussed above, the nuclease of Mre11 is largely dispensable for the processing of clean DSBs, yet MRX was shown to have a structural role in promoting the resection by Sgs1--Dna2. In particular, Mre11 interacts with Sgs1 and stimulates its helicase activity (27,28,80). As the MRX complex localizes very early to DSBs (25), it has been pro-posed that it might recruit Sgs1--Dna2 to DNA ends (81). Furthermore, the Sgs1 hel-icase is known to form a complex with Top3--Rmi1 (72,82). Surprisingly both Top3 and Rmi1 were found to be required for DNA end resection by Sgs1 and Dna2 in vivo (9) as well as in vitro, independently of the topoisomerase activity of Top3 (27). The heterodimer strongly stimulates the Sgs1 helicase, which is especially apparent under physiological salt concentrations (27,28). The mechanism by which Top3--Rmi1 pro-mote DNA unwinding by Sgs1 is not yet clear, though it is obvious that Sgs1--Top3--Rmi1 form a very integrated functional complex (82,83). Additionally, Sgs1 was de-scribed to interact with Rad51 (84). The functional significance of this interaction is not yet clear, however it is attractive to hy-pothesize that it might help loading Rad51 directly on resected ssDNA in analogous fashion to RecBCD--or RecFOR--mediated loading of RecA (17).
The mechanism of DNA end resection by Sgs1 and Dna2 is conserved in evolution. Human DNA2 forms a complex with the human Sgs1 homolog, the Bloom (BLM) hel-icase, and the resection by DNA2--BLM is similarly promoted by the human RPA, MRN and TopoIIIα--RMI1--RMI2 proteins (85,86). In addition, DNA2 also interacts with anoth-er RecQ family helicase, Werner (WRN). Al-so BLM--WRN complex promotes resection in vivo and in vitro, showing a functional re-dundancy in DSB processing in human cells (87).

Exo1 resection pathway
Unlike the Dna2 nuclease that is specific for ssDNA, the nuclease activity of exonu-clease 1 (Exo1) degrades 5'--terminated strand within dsDNA (88). Therefore, Exo1 does not require a helicase partner to un-wind DNA, and directly produces the re-quired 3'--tailed DNA (37,88) (Fig. 3b). In humans, the BLM protein was found to stimulate resection by EXO1 in a helicase-independent manner, but a similar mecha-nism was not detected in yeast (9,22,23,37,89,90). Before Exo1's role in DNA end resection was discovered, Exo1 had been known to play an important function in the postrepli-cative mismatch repair. Reconstitution ex-periments revealed that Exo1 nuclease is rather distributive and requires the support of the mismatch recognition complex MutSα to stimulate its processivity in the presence of a mismatch (91). Similarly, various fac-tors were identified that promote the Exo1 nuclease in resection. As in the case of the Sgs1--Dna2 pathway, the MRX complex pro-vides a structural role to stimulate Exo1 (37,81), which is further enhanced by Sae2 (29). However, efficient Exo1--dependent resection occurred even in the absence of the MRX complex in vivo, suggesting that other factors may promote the Exo1 nucle-ase (9,23). That may include the ssDNA binding proteins RPA or the Sensor of ssD--NA complex 1 (SOSS1) (37,92,93). Further--more, the 9--1--1 clamp was also found to promote long--range resection independent-ly of its checkpoint signaling activity under certain conditions (94), which is conserved in human cells (95). Finally, PCNA was found to promote human EXO1 processivity by enhancing its association with DNA (85,96).
Acknowledgement: I would like to thank to Alessandro Sar-tori, Elda Cannavo, Lucie Mlejnkova, Cosimo Pinto, Maryna Levikova, Lepakshi Ranjha and Roopesh Anand (all University of Zur-ich) for discussions and comments on the manuscript. This work was supported by Swiss National Science Foundation Grant (PP00P3 133636). I apologize to colleagues whose work could not be discussed here due to space limitation. REFERENCES:  The Mre11--Rad50--Xrs2 (MRX) complex is rapidly recruited to DNA ends upon break formation. The nuclease activity of Mre11 is not required for resection, but the MRX complex has a role to recruit components of the processive pathways that include either Sgs1--Dna2 or Exo1. In some cases the structural role of the MRX complex can be bypassed. DNA is subsequently resected by either Sgs1--Dna2 or Exo1 in a proces-sive manner. Only a monomer of MRX is depicted for clarity reasons. b, Resection of blocked (dirty) DNA ends. The MRX complex is rapidly recruited to DNA ends, which is followed by Sae2. The nuclease activity of Mre11 is required, and it cleaves endonucleo-lytically the 5'--terminated DNA strand away from the end in a reaction stimulated by phosphorylated Sae2. Furthermore, MRX also likely recruits Sgs1--Dna2 or Exo1 to the endonuclease cut site. The endonuclease cut site provides an entry point for the Sgs1--Dna2 or Exo1 nucleases, which carry out long--range resection. The exonuclease of Mre11 then might degrade DNA in a 3' → 5' direction back towards the DNA break. Figure 3. Mechanism of long--range DNA end resection by Sgs1--Dna2 or Exo1 pathways. a, DNA end resection by Sgs1--Dna2. Sgs1 translocates with a 3' → 5' polarity on one DNA strand and unwinds DNA. Unwound ssDNA is coated by RPA, which directs the nucleolytic activity of Dna2 towards the 5'--terminated DNA strand. Whether the 5' → 3' motor activity of Dna2 participates in DNA end resection to form a bidirectional hel-icase remains to be demonstrated. b, DNA end resection by Exo1. The Exo1 nuclease is specific for dsDNA and has a 5' → 3' polarity, which directly results in 3' tailed DNA.     Figure 3