Processing of homologous recombination repair intermediates by the ...

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Cell Cycle 10:18, 3078-3085; September 15, 2011; © 2011 Landes Bioscience

Processing of homologous recombination repair intermediates by the Sgs1-Top3-Rmi1 and Mus81-Mms4 complexes Ian D. Hickson and Hocine W. Mankouri* Nordea Center for Healthy Aging; Department of Cellular and Molecular Medicine; University of Copenhagen; Copenhagen, Denmark

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omologous recombination repair (HRR) is an evolutionarily conserved cellular process that is important for the maintenance of genome stability during S phase. Inactivation of the Saccharomyces cerevisiae Sgs1-Top3Rmi1 complex leads to the accumulation of unprocessed, X-shaped HRR intermediates (X structures) following replicative stress. Further characterization of these X structures may reveal why loss of BLM (the human Sgs1 ortholog) leads to the human cancer predisposition disorder, Bloom syndrome. In two recent complementary studies, we examined the nature of the X structures arising in yeast strains lacking Sgs1, Top3 or Rmi1 by identifying which proteins could process these structures in vivo. We revealed that the unprocessed X structures that accumulate in these strains could be resolved by the ectopic overexpression of two different Holliday junction (HJ) resolvases, and that the endogenous Mus81-Mms4 endonuclease could also remove them, albeit slowly. In this review, we discuss the implications of these results and review the putative roles for the Sgs1Top3-Rmi1 and Mus81-Mms4 complexes in the processing of various types of HRR intermediates during S phase.

RECQL4, RECQ5 and WRN. These proteins have received considerable interest due to their putative roles in the suppression of cancer and premature aging in humans. Mutations in BLM cause Bloom’s syndrome, which is associated with increased cancer predisposition, whereas mutations in WRN or RECQ4 cause distinct disorders characterized by premature aging and developmental abnormalities. More specifically, mutations in WRN cause Werner syndrome, and mutations in RECQ4 cause Baller-Gerold syndrome, RAPADILINO syndrome or RothmundThomson syndrome. Although no heritable diseases have yet to be associated with RECQ1 or RECQ5 deficiency, mutation of these RecQ helicases may affect cancer predisposition or survival rates.1,2 In Saccharomyces cerevisiae (budding yeast), there is one RecQ helicase, called Sgs1. Therefore, analysis of RecQ helicase functions and genetic interactions is greatly simplified in this organism.3 Sgs1 is thought to be particularly pertinent to the study of BLM, because Sgs1 shares a number of features with BLM that are not observed with the other human RecQ helicases. First, BLM is the only human RecQ helicase that shares a similar protein domain architecture to that of Sgs1. The other four human RecQ helicases appear to have lost and/or acquired additional protein domains throughout the course of evolution. Second, BLM and Sgs1 exist in a complex with an evolutionarily conserved set of proteins. BLM and Sgs1 (and also E. coli RecQ) associate with and stimulate the activity of a type IA topoisomerase (Top3 in yeast; hTOPOIIIα in humans) that can modify the topological status

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Key words: RusA, GEN1, Eme1, RecQ, topoisomerase Abbreviations: BS, Bloom’s syndrome; CO, crossover; DSB, double-strand break; HRR, homologous recombination repair; HJ, Holliday junction; MMS, methylmethanesulfonate; NJ, nicked junction; RTR, RecQTopoisomerase‑Rmi Submitted: 06/13/11 Revised: 07/21/11 Accepted: 07/21/11 DOI: 10.4161/cc.10.18.16919 *Correspondence to: Hocine W. Mankouri; Email: [email protected]

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Introduction The RecQ proteins form an evolutionarily conserved family of DNA helicases that are important for the maintenance of genome stability. The family is named after the “prototypic” member, E. coli RecQ. Five RecQ helicases have been identified in humans: BLM, RECQ1,

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of DNA by passing one single strand of DNA through the other.4-14 Additionally, Sgs1-Top3 and BLM-hTOPOIIIα associate with conserved OB fold-containing proteins (Rmi1 in yeast; BLAP75/hRMI1 and BLAP18/RMI2 in humans), which appear to stimulate the enzymatic functions of Sgs1-Top3 and BLM-hTOPOIIIα in vitro.14-24 Current thinking suggests that the Sgs1-Top3-Rmi1 and BLMhTOPOIIIα-RMI1-RMI2 complexes [henceforth denoted as the “RTR (RecQTopoisomerase-Rmi) complexes”] act cooperatively as a evolutionarily conserved “dissolvasome” that can process different types of DNA structures that arise during DNA replication, homologous recombination repair (HRR) and mitosis.25,26 The S. cerevisiae and human RTR complexes have both been strongly implicated in HRR. This is an important cellular process that allows cells to copy genetic information from a homologous sequence and accurately repair DNA breaks and ssDNA gaps. These types of lesions can arise due to DNA damage or due to discontinuities or errors in DNA replication. Evidence that these proteins act in HRR is suggested by the observations that inactivation of components of the S. cerevisiae or human RTR complexes causes elevated levels of mitotic recombination, sister chromatid/chromosome exchanges and genome instability.26 Indeed, sister chromatid exchanges are the defining feature (and a diagnostic marker) of BS in human cells.27 Interestingly, unprocessed HRR intermediates are detectable in extracts from yeast strains impaired for RTR components following replication stress (using the DNA-alkylating agent, MMS). These structures were revealed using two-dimensional (2D) gel electrophoresis, a technique that allows the detection of various DNA structures such as replication origins (“bubbles”), replication forks (Y-structures) and joint molecules (X structures) at a precise region of the genome at any given time during S phase.28 Inactivation of any single RTR component in yeast leads to the accumulation of postreplicative, X-shaped DNA structures that are dependent upon the HRR machinery for their accumulation.29-31 We propose, therefore, that these X structures represent an unprocessed intermediate that is

formed during HRR-mediated repair of ssDNA gaps that are left behind ongoing replication forks. However, the precise nature of these X-shaped DNA structures has remained undefined, and is the subject of some debate.32 Although 2D gel electrophoresis represents a powerful tool for the spatiotemporal detection of locus-specific DNA structures in vivo, a limitation of the technique is that the precise nature of X-shaped DNA structures cannot be determined directly. Characterization of Unprocessed HRR Intermediates Arising in Cells Impaired for RTR Function In two recent complementary studies, we examined the nature of the MMS-induced X structures arising in cells impaired for RTR components.33,34 This was achieved by deleting SGS1 or conditionally inactivating either TOP3 (by overexpressing a dominant-negative allele, TOP3Y356F ) or RMI1 (using a temperature-sensitive mutant, rmi1-1). We then sought to identify proteins that could resolve the MMS-induced X structures in vivo. Using this approach, we demonstrated that the ectopic overexpression of two evolutionarily divergent Holliday junction (HJ) resolvases, E. coli RusA and a constitutively active fragment of human GEN1, GEN11‑527, could diminish the level of MMS-induced X structures that normally persist in S. cerevisiae sgs1, rmi1-1 and TOP3Y356F strains.33,34 A shared feature of these proteins is that they introduce symmetrically related nicks into two strands of the same polarity at the junction crossover in covalently closed four-way junctions.35-40 This suggests that the MMS-induced X structures detected in RTR-defective strains either comprise or are converted into HJ-containing structures between sister chromatids. Interestingly, unprocessed HJs arising in Sgs1-defective strains could also be eliminated by the reactivation of Sgs1 in vivo, suggesting that the RTR complex is able to directly process HJ-containing HRR intermediates in vivo once formed.34 However, we note that this does not exclude the possibility that the RTR complex also prevents HJ formation in wild-type cells.

We also examined which endogenous yeast proteins are required for the resolution of unprocessed HRR intermediates in the absence of a functional RTR complex. An interesting feature of the X structures arising in RTR-defective strains is that they accumulate rapidly during a perturbed S phase and then disappear slowly upon the removal of MMS.41 This suggests that other proteins exist in vivo that can also process these structures, albeit with apparently slower kinetics. Based on our hypothesis that X structures comprise or are converted into HJ-containing DNA structures, we examined if any putative yeast HJ-cleavage enzymes were required for X structure disappearance in RTRdefective strains. The contribution of three putative HJ-processing activities was examined: Mus81-Mms4, Slx1–Slx4 and Yen1. Each of these proteins (and/or their homologs) has previously been implicated in the processing of HJs to some degree.42 Because loss of Mus81-Mms4 or Slx1– Slx4 is synthetically lethal when combined with mutation of any RTR components, we examined the consequences of deleting these putative HJ resolvases in the conditional rmi1-1 and TOP3Y356F strains. Interestingly, we demonstrated that the Mus81-Mms4 endonuclease, but not Slx1-Slx4 or Yen1, comprises the primary X structure-processing activity identified in rmi1-1 and TOP3Y356F mutants.33 We observed that loss of Mus81-Mms4 did not by itself cause the formation of excess MMS-induced X structures, even when combined with impairment of Top3 or Rmi1. Rather, loss of Mus81-Mms4 led to an apparent defect in X-structure resolution in strains that were impaired for Top3 or Rmi1.33 We propose, therefore, that the RTR complex constitutes the main pathway for the turnover of MMS-induced X structures, but that Mus81-Mms4 can also resolve these intermediates in the absence of a functional RTR complex. In addition to characterizing an endogenous substrate that can be processed by the RTR complex or Mus81-Mms4 in vivo, we suggest that our data also explains why it has been reported previously that expression of ectopic HJ resolvases can suppress various RecQ helicase and Mus81-Mms4/ Eme1 deficiencies in S. cerevisiae, S. pombe and human cells.35,43-51


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What is the Functional Relationship between the RTR Complex and the Mus81-Mms4 Endonuclease? Our findings that Mus81-Mms4 can process unresolved HRR intermediates that arise in RTR-defective strains during S phase is reminiscent of the reported findings in Saccharomyces cerevisiae meiotic cells. In these cells, Sgs1 largely acts to limit the formation of HRR intermediates that can be resolved by Mus81-Mms4.52-55 However, it should be noted that meiotic HRR is initiated by genome-wide programmed double-strand breaks (DSBs) catalyzed by the transesterase Spo11 and is influenced by meiosis-specific (ZMM) proteins.53,54,56 Moreover, HJ formation is a prerequisite for homologous chromosome pairing during meiosis. Therefore, meiotic and mitotic cells have fundamentally different requirements for HRR, and this probably influences precisely which types of HRR intermediates form, and how they are resolved. Nevertheless, there are clear similarities between these studies regarding the functional relationship between the RTR complex and Mus81-Mms4. In the remainder of this review, we discuss our hypotheses regarding the precise nature of the unprocessed HRR intermediates in RTR-defective strains and propose a model describing the putative roles of the RTR complex and Mus81Mms4 endonuclease in mitotic cells. In attempting to reconcile our findings with the available biochemical data, we propose that the unprocessed HRR intermediates we detect could comprise one, or possibly a mixture, of three distinct types of DNA structures: single HJs, nicked junctions and double HJs. These would appear qualitatively similar on 2D gels, and we will therefore discuss each of these DNA structures in turn. For brevity, and because the X structures we detect on 2D gels appear to persist in replicated DNA fragments (i.e., behind replication forks), we will limit our discussion to junction-containing structures that arise as intermediates during post-replicative filling of ssDNA gaps by HRR. These structures probably form when DNA lesions are bypassed during DNA replication or when lagging strand DNA synthesis is perturbed.

Proposal 1: X Structures Contain Single HJs Single HJs (sHJs) contain a (cross-strand) 4-way junction and arise during HRR once Rad51-mediated 3' strand invasion occurs (Fig. 1i). This structure could be cleaved by HJ resolvases or branch migrated by a RecQ helicases.57-59 Although a role for S. cerevisiae Mus81-Mms4, S. pombe Mus81-Eme1 and human MUS81-EME1 (collectively referred to as “the Mus81 complex”) in cleaving HJs has been proposed previously in reference 60 and 61, the preferred substrate is a nicked or unligated junction (discussed in proposal 2 below).42 However, it is worth noting that the activity attributable to Mus81-Eme1 from S. pombe cellular extracts exhibits a more robust HJ cleavage ability than does recombinant Mus81-Eme1.62 This suggests that an as-yet-uncharacterized cellular factor or post-translational modification may enhance the putative HJ cleavage activity of Mus81-Eme1 in vivo. Further evidence for an in vivo role for the Mus81 complex in HJ cleavage comes from genetic analyses in S. cerevisiae and S. pombe. In particular, the Mus81 complex appears to control most, or a significant fraction of, meiotic crossover (CO) events in S. pombe and S. cerevisiae, respectively, as would be predicted for a HJ resolution enzyme.42 Furthermore, sHJs have also been detected as meiotic intermediates in S. pombe mus81 mutants.63 Mus81 is also required for the majority of COs following DSB induction during mitosis in S. cerevisiae, with Yen1 apparently acting as “backup” activity.64 Although the exact nature of the structure(s) processed by Mus81 (or Yen1) during mitotic DSB repair were not determined, the authors proposed that an unresolved sHJ in mus81 yen1 double mutants could cause chromosome breakage during mitosis, which results in the observed increase in POL32dependent, break-induced replication.64 Mus81 is also required for the resolution of artificial plasmid-based structures in S. cerevisiae that resemble, or form, sHJs in vivo.48,65 Furthermore, in one of these studies, the mus81 defect could also be complemented by the expression of the HJ resolvase RusA.48 Indeed, increased or ectopic, expression of HJ resolvases (RusA,

Yen1 or GEN11-527) has been demonstrated to suppress various phenotypes caused by Mus81 complex deficiencies in S. cerevisiae, S. pombe and human cell lines.35,44‑51 Taken together with our data, these findings are consistent with two possible models: (1) HJs are an in vivo substrate for the Mus81 complex or (2) HJs can be converted into a DNA structure that can be cleaved by the Mus81 complex. Proposal 2: X Structures Contain Nicked Junctions Although the Mus81 complex can cleave HJs in vitro, this DNA structure does not appear to be the preferred substrate for the Mus81 complex.60,61 Rather, the Mus81 complex from S. cerevisiae, S. pombe and human cells prefers branched (three- or four-way) substrates that contain a discontinuity or nick adjacent to the branchpoint of the junction.42,60,61,66,67 This is believed to be due to the greater flexibility of the arms of the nicked substrates, allowing the incision point to be positioned into the catalytic site of the Mus81 complex.68,69 For this reason, the Mus81 complex is sometimes referred to as a “nicked-junction endonuclease.” 70 In the model for ssDNA gap repair in Figure 1, we demonstrate two possible ways that a structure that resembles an unligated or nicked junction (NJ; Fig. 1ii) can form. First, maturation of the 3' invading strand creates a NJ-like structure immediately prior to second-end capture and the formation of a covalently closed dHJ structure (discussed in proposal 3 below). Another, albeit speculative, possibility is that intact HJs could be converted into NJs by an as-yet-uncharacterized HJ “nickase.” Do these data suggest that the X structures we detect in RTR-defective strains comprise NJs? It remains an untested possibility. Although RusA can cleave NJs in vitro, it is likely that this reaction requires branch migration of the junction point to a preferred cleavage site.71 Therefore, we do not favor the idea that the X structures we detect comprise immobile NJs. One intriguing possibly is that branch migration of an unprocessed sHJ in RTR-defective strains to a DNA gap/nick could then promote NJ cleavage by the Mus81 complex


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Figure 1. Proposed model describing the roles of the RTR and Mus81 complexes in the resolution of HRR intermediates. MMS-induced DNA lesions cause discontinuous DNA synthesis, leading to the accumulation of post-replicative ssDNA gaps. These ssDNA gaps can be repaired by Rad51dependent homologous recombination repair (HRR). Three key DNA structures discussed in this review that may form as intermediates in HRR are indicated in gray boxes: (i) single Holliday junctions (sHJs, magneta outline), (ii) nicked junctions (NJs, green outline) and (iii) double Holliday junctions (dHJs, cyan outline). The RTR (RecQ-Topoisomerase-Rmi) complex may act at several stages to limit the formation or promote the resolution, of certain HRR intermediates. When the RTR complex is impaired, unprocessed HRR intermediates persist that contain or are interconverted into DNA structures that contain one or more Holliday junctions (HJs). A single HJ (sHJ) arises after Rad51-mediated strand invasion occurs (i). The Mus81 complex (Mus81Mms4) can process unresolved HRR intermediates in the absence of a functional RTR complex, probably by cleaving nicked junction (NJ) intermediates (ii). However, another possibility is that the Mus81 complex associates with an additional cellular factor(s) in vivo that allows it to cleave fully ligated HJs. Together, the RTR and Mus81 complexes may act (possibly cooperatively) to limit the formation of double HJs (dHJs) during HRR (iii). In doing so, these enzymes may limit a potentially harmful source of genome rearrangements that can occur due to the uncoordinated endonucleolytic processing of dHJs by HJ resolvases. If dHJs do form, the RTR complex can also remove them (without any genome rearrangements occurring) by catalyzing dHJ dissolution.


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(Fig. 1). This could explain the apparently “slower” Mus81-Mms4-dependent process we detect, because the rate-limiting step of X-structure resolution would depend on the branch migration of sHJs to the position of a DNA nick. Interestingly, S. cerevisiae Mus81 and human MUS81 interact with a DNA translocase, Rad54 and RAD54, respectively, which can catalyze branch migration of four-way junctions in vitro.72-77 Furthermore, Mus81/ MUS81 endonucleotyic activity is stimulated by Rad54/RAD54, suggesting these proteins could cooperate in the processing of HRR intermediates.74,75 Interestingly, human BLM can also stimulate MUS81EME1 endonuclease activity in vitro, suggesting cooperativity between these proteins in some circumstances.78 Taken together, these data are consistent with the Mus81 complex having a cooperative role with proteins that can catalyze branch migration of HJs in the resolution of HRR intermediates.

both the S. cerevisiae and human RTR complexes, suggesting that this function of the RTR complex is evolutionarily conserved.12,18,19,21,24 The other way in which a dHJ intermediate can be processed is by the endonucleotytic cleavage by a HJ resolvase and religation of individual HJs (Fig. 1). Depending on the relative orientation of cleavage and religation, this can lead either to crossovers (COs) or noncrossovers (NCOs). Although COs are unlikely to be deleterious between sister chromatids, they may cause potentially harmful chromosomal rearrangements if they arise between non-homologous sequences. Although dHJs have been detected as intermediates of DSB repair in both meiotic and mitotic cells in vivo, they appear to form at a 10-fold lower frequency (per DSB) in mitotic cells than they do in meiotic cells.79,80 One possibility, therefore, is that mitotic cells normally attempt to limit dHJ formation during HRR of ssDNA gaps. This could be achieved by the combined (and possibly collaborative) activities of the RTR complex (which disrupts early HRR intermediates at the initial strand invasion or sHJ stage) and the Mus81 complex (which cuts NJs prior to second-end capture). Interestingly, a defect in mitotic dHJ turnover during DSB repair was detected in sgs1 mutants,80 although it remains to be determined if loss of the Mus81 complex (either alone or in conjunction with RTR impairment) also affects the turnover of mitotic dHJs in this system. It is also worth noting that the assays used to characterize meiotic and mitotic dHJs could not determine whether these structures are fully ligated (i.e., do not contain any nicks) or not.42 Although the RTR and Mus81 complexes may principally act to limit dHJ formation in mitotic cells, another intriguing possibility is that these complexes may comprise two parallel processes for the elimination of dHJs once they have formed (Fig. 1). If true, this suggests that the Mus81 complex is a bona fide HJ cleavage enzyme that can act upon fully ligated dHJ intermediates in vivo. Interestingly, the increased SCEs observed in BS cells are reduced by the depletion of MUS81, suggesting that the processing of

unresolved DNA structures by MUS81 is responsible for elevated SCEs in BS cells.81 However, whether the putative unresolved DNA structures arising in BS cells comprise unprocessed dHJs remains to be determined. Furthermore, the Mus81 complex can generate COs before HRR intermediates mature into dHJs when the initiating lesion is a DSB (rather than a ssDNA gap, as depicted in Fig. 1).71 Perspectives Although we have proposed three possibilities to explain what the MMSinduced X structures are, we note that the ideas proposed here are not mutually exclusive, and the RTR and Mus81 complexes could act on sHJs, NJs and dHJs in vivo. Indeed, both sHJ and dHJs accumulate in S. cerevisiae meiotic cells lacking Sgs1 and Mms4.55 Furthermore, these structures may be to some degree interconvertable in vivo (Fig. 1). One interesting possibility is also that the preferred substrate specificities of the RTR and Mus81 complexes could vary throughout the cell cycle. For example, it has been proposed that the preference of the Mus81 complex for nicked vs. intact HJs may depend on its dimerization state, post-translational modifications and additional cellular cofactors.60-62 Therefore, it is conceivable that the apparently “slower” activity of the Mus81 complex reflects a (cell cycle checkpoint-mediated) modification of Mus81-Mms4 activity in late-S phase or G2 . Our characterization of an in vivo substrate for the Mus81 complex should greatly facilitate functional analyses of the Mus81 complex in future studies. A number of other key questions remain. First, which other proteins are involved in X structure processing when the RTR complex is impaired, and do these act in concert with the Mus81 complex, or do they define a distinct process? For example, if the Mus81 complex has an additional co-activator(s) that permits it to function as a bona fide HJ cleavage enzyme in vivo, then the identification of this factor awaits characterization. Second, the functions of the RTR and Mus81 complexes are probably not confined to resolving HRR intermediates,


Proposal 3: X Structures Contain Double HJs

If second-end capture of the invading 3' end occurs, then an intermediate known as a double HJ (dHJ; Fig. 1iii) forms. Based on the known enzymology of the Mus81 complex, one proposed role of this complex is to cut NJs prior to second-end capture so as to prevent the formation of this intermediate (Fig. 1). A dHJ can be considered distinct from a sHJ or NJ, because it comprises a topologically linked and covalently closed DNA structure that can be processed by the RTR complex in a process known as dHJ dissolution.12 In this, one or both HJs are branch migrated toward each other until they merge to form a hemicatenane, which can be decatenated by the activity of the type IA topoisomerases (Fig. 1). It is likely that the RTR complex acts cooperatively in this process: the RecQ helicase (Sgs1/BLM) catalyzes convergent branch migration, with the type IA topoisomerase (Top3/ hTOPOIIIα) relieving the concomitant torsional stress, to produce the hemicatenane intermediate that is ultimately resolved by Top3/hTOPOIIIα acting in conjunction with Rmi1/RMI1-RMI2. This process has been demonstrated for

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and these proteins probably also process other types of DNA structures that arise during DNA replication and mitosis.82 For example, in addition to the HRR intermediates discussed in this review, the Mus81 and RTR complexes also likely perform important functions at stalled RFs that arise during DNA replication.83 Therefore, it will be of great interest to identify additional in vivo substrates for the RTR and Mus81 complexes, outside of HRR. Finally, what are the in vivo roles of the other putative yeast HJ resolvases, such as Slx1-Slx4 and Yen1? Although our analyses have not yet revealed a clear role for these proteins in the processing of HRR intermediates in vivo, loss of Slx1– Slx4 causes synthetic lethality and an aberrant mitosis in the rmi1-1 strain, and yen1 mus81 double mutants exhibit synergistic growth defects.33,49,64,84,85 Because sgs1slx1 and sgs1slx4 synthetic lethality cannot be suppressed by the abolition of HRR, this suggests that Slx1–Slx4 may process HJ-containing structures that arise outside of HRR, such as regressed replication forks.47,85 The role of Yen1 also remains enigmatic and may only become apparent when the Mus81 complex is compromised.49,64 However, because the abolition of HRR can alleviate mus81yen1 phenotypes, this suggests that Yen1 may resolve (otherwise toxic) HRR intermediates, at least under some circumstances.49 Our future studies will thus be aimed at identifying the in vivo substrates for Slx1– Slx4 and Yen1. Clearly this has important relevance to human cells, because combined siRNA-mediated depletion of MUS81 and GEN1, or SLX4 and GEN1, causes severe chromosome abnormalities in human BS cells.81 Furthermore, SLX4 mutations have recently been identified in a new subtype of Fanconi anemia (FANCP), suggesting that defective HJ processing may contribute to the etiology of this disease.86,87 Additionally, MUS81 downregulation has recently been reported for a number of tumor types, and this may be a novel prognostic marker for certain cancer types.88-90 Therefore, elucidating the in vivo functions, relationship between and regulation of the various HJ processing activities that exist in eukaryotic cells could have important implications for the development of new

prognostic, diagnostic or therapeutic tools for human diseases. Acknowledgments

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