AtGEN1 and AtSEND1, Two Paralogs in Arabidopsis,Possess Holliday Junction Resolvase Activity

Holliday junctions (HJs) are physical links between homologous DNA molecules that arise as central intermediary structures during homologous recombination and repair in meiotic and somatic cells. It is necessary for these structures to be resolved to ensure correct chromosome segregation and other functions. In eukaryotes, including plants, homologs of a gene called XPG-like endonuclease1 (GEN1) have been identified that process HJs in a manner analogous to the HJ resolvases of phages, archaea, and bacteria. Here, we report that Arabidopsis (Arabidopsis thaliana), a eukaryotic organism, has two functional GEN1 homologs instead of one. Like all known eukaryotic resolvases, AtGEN1 and Arabidopsis single-strand DNA endonuclease1 both belong to class IV of the Rad2/XPG family of nucleases. Their resolvase activity shares the characteristics of the Escherichia coli radiation and UV sensitive C paradigm for resolvases, which involves resolving HJs by symmetrically oriented incisions in two opposing strands. This leads to ligatable products without the need for further processing. The observation that the sequence context influences the cleavage by the enzymes can be interpreted as a hint for the existence of sequence specificity. The two Arabidopsis paralogs differ in their preferred sequences. The precise cleavage positions observed for the resolution of mobile nicked HJs suggest that these cleavage positions are determined by both the substrate structure and the sequence context at the junction point.To counter the effects of endogenous and exogenous factors that threaten the genome integrity, efficient mechanisms have evolved to ensure the faithful transmission of genetic information (Tuteja et al., 2001). Double-strand breaks, induced by conditions such as ionizing radiation or replication fork (RF) stalling, are among the most deleterious lesions (Jackson and Bartek, 2009). To protect the genome from consequences of these lesions, the cells have ancient double-strand break repair mechanisms, including the homologous recombination (HR) pathway. The HR mechanism is also of great importance in the intentional genetic recombination during sexual reproduction. A key intermediate in HR is the so-called Holliday junction (HJ), a structure that was first suggested in the context of a gene conversion model in fungi (Holliday, 1964) and later shown to arise in somatic and meiotic cells (Szostak et al., 1983; Schwacha and Kleckner, 1995; Cromie et al., 2006; Bzymek et al., 2010).HJs are structures consisting of four DNA strands of two homologous DNA helices (e.g. homologous chromosomes or sister chromatids). They arise through invasion of one single strand from each of two helices into the other double strand. This results in two continuous strands (one per helix) and two strands that cross from one helix into the other. Schematics often depict the HJs with a parallel orientation of the helices, in which the crossing strands cross each other as was originally postulated (Holliday, 1964). However, HJs based on oligonucleotides have been shown to adopt an antiparallel conformation (for review, see Lilley, 2000). In this configuration, the junction resembles the letter H in a lateral view, and the crossing strands actually perform U turns. The crossing strands represent physical links between the two DNA strands involved. If a RF is restored by HR-mediated repair during mitosis, the resulting HJ usually involves the two sister chromatids of one chromosome (Li and Heyer, 2008). In meiosis, the physical links in the shape of HJs arise because of meiotic crossover between homologous chromosomes. In either case, these links must be resolved to ensure unperturbed cell survival.The importance of resolving the HJs for the survival of cells and organisms is highlighted by the phenotypes described for mutants defective for the known pathways of HJ resolution. One of these pathways is the resolution by canonical HJ resolvases, enzymes that cleave the two opposing strands of a HJ in perfectly symmetric positions relative to the junction point, which results in readily ligatable nicked duplex (nD) products (Svendsen and Harper, 2010). This property distinguishes the canonical HJ resolvases from the noncanonical resolvases (see below).The main resolvase of Escherichia coli is radiation and UV sensitive C (RuvC), which is part of the E. coli resolvasome (RuvABC complex; Otsuji et al., 1974; Sharples et al., 1990, 1999). In this complex, a HJ is sandwiched between two RuvA tetramers (Panyutin and Hsieh, 1994). Two RuvB complexes form ATP-dependent motors of branch migration, with two opposing helical arms of the junction threaded through their central openings. For the resolution of the HJ, one RuvA tetramer is replaced by a RuvC homodimer. This homodimer positions two active sites at the center of the junction that are poised to cleave the junction point if a preferred consensus sequence of the form 5′-(A/T)TT^↓(G/C)-3′ is encountered. The requirement for this correct sequence is quite strict; even a single base change can lead to a drastic reduction of the cleavage efficiency (Shah et al., 1994). Isolated EcRuvC is also active in vitro and binds only HJ structures with high specificity. This binding is independent of the sequence context, but the cleavage depends on the specific sequence (Iwasaki et al., 1991; Benson and West, 1994; Dunderdale et al., 1994). The exact cleavage position has been determined to be either one nucleotide 3′ or 5′ from the junction or at the junction point (Bennett and West, 1996; Shida et al., 1996; Osman et al., 2009). The well-characterized EcRuvC is often referred to as a paradigm of canonical HJ resolution.Eukaryotes have evolved a more complex interplay of different HJ resolution pathways (Schwartz and Heyer, 2011; Zakharyevich et al., 2012). A defined complex, consisting of a recombination deficiency Q (RecQ) helicase (AtRECQ4A in Arabidopsis [Arabidopsis thaliana], Bloom syndrome protein in human, and Slow growth suppression1 (Sgs1) in yeast [Saccharomyces cerevisiae]), a type IA topoisomerase (DNA topoisomerase 3-alpha [TOP3A] in Arabidopsis, HsTOPOIIIα in human, and ScTop3 in yeast), and the structural protein RecQ-mediated genome instability1 (AtRMI1 in Arabidopsis, HsRMI1 in human, and ScRmi1 in yeast; RTR complex), mediates the so-called dissolution pathway. The crossing points of a double HJ are brought together by branch migration catalyzed by the helicase followed by decatenation catalyzed by the topoisomerase (Wu and Hickson, 2003; Hartung et al., 2007a, 2008; Mankouri and Hickson, 2007; Yang et al., 2010). In addition to the catalytic activities, a functional RTR complex also requires structural functions based on protein-protein interactions, for which RMI1 plays an essential role (Mullen et al., 2005; Chen and Brill, 2007; Bonnet et al., 2013; Schröpfer et al., 2014). Dissolution leads to noncross-over products and therefore, is a major mechanism in somatic yeast cells (Gangloff et al., 1994; Ira et al., 2003; Matos et al., 2011). In Arabidopsis, the loss of RTR component function leads to elevated rates of HR as well as sensitivity to UV light and methylmethane sulfonate (MMS; Bagherieh-Najjar et al., 2005; Hartung et al., 2007a; Bonnet et al., 2013). Mutants of AtRMI1 and AtTOP3A exhibit severe and unique meiotic phenotypes (Chelysheva et al., 2008; Hartung et al., 2008). This meiosis I arrest is dependent on HR, but the exact nature of the recombination intermediates that are involved remains unclear (Li et al., 2004; Hartung et al., 2007b; Knoll et al., 2014).Dissolution acts in parallel with a second pathway mediated by the structure-specific endonuclease MMS and UV-sensitive protein81 (MUS81) as shown by the fact that the additional mutation of ScSgs1/AtRECQ4A leads to synthetic lethality (Mullen et al., 2001; Hartung et al., 2006; Mannuss et al., 2010). Single mutants of MUS81 in yeast, human, Drosophila melanogaster, and Arabidopsis are sensitive to DNA-damaging agents that perturb RFs and show reduced HR after induction of double-strand breaks (Boddy et al., 2001; Hanada et al., 2006; Hartung et al., 2006). The MUS81 homologs form heterodimers with the noncatalytic subunit essential meiotic endonuclease1 (EME1; ScMms4 in S. cerevisiae). SpMus81-Eme1 was, to our knowledge, the first nuclear endonuclease reported to be capable of resolving HJs (Boddy et al., 2001). The Arabidopsis complexes can be formed with the two different subunits: AtEME1A or AtEME1B (Geuting et al., 2009). AtMUS81-EME1A/B, like the fission yeast ortholog, preferentially cleaves nicked Holliday junctions (nHJs) and 3′-flaps but also shows weaker activity on intact HJs in vitro (Boddy et al., 2001; Osman et al., 2003; Geuting et al., 2009; Schwartz and Heyer, 2011). MUS81 homologs are key players in meiotic cross-over generation (Osman et al., 2003; Berchowitz et al., 2007; Higgins et al., 2008). Although cross-over formation is solely dependent on SpMus81 in fission yeast, this function was shown to be shared with ScYen1 in budding yeast (Osman et al., 2003; Blanco et al., 2010; Ho et al., 2010; Tay and Wu, 2010). Tightly regulated by cell division cycle5-dependent hyperphosphorylation at the end of prophase I, the main activity of ScMus81-Mms4 is timed to coordinate with the formation of chiasmata and HJs that link the homologous chromosomes. This role in meiosis I is shown by the failure of chromosome segregation at the end of meiosis I in ScMus81 mutants (Matos et al., 2011). Interestingly, the chromosomes could be segregated at the end of meiosis II because of the presence of ScYen1. In contrast to canonical HJ resolvases, the hallmark of the MUS81-EME1 cleavage mechanism is the asymmetry of the second incision relative to either a first incision or a preexisting nick. This difference classifies MUS81-EME1 as a noncanonical resolvase. Its products need additional processing by gap-filling or flap-cleaving enzymes to allow religation (Boddy et al., 2001; Geuting et al., 2009).In very recent studies, HsMUS81-EME1 was found to constitute an essential canonical HJ resolvase with HsSLX1-SLX4 (SLX for synthetic lethal of unknown function), in which a first incision is made by HsSLX1-SLX4 followed by the enhanced action of the HsMUS81-EME1 subunits on the resulting nHJ (Garner et al., 2013; Wyatt et al., 2013). HsSLX1-SLX4 had previously been described as a canonical resolvase, albeit producing only a low level of symmetrically cut ligatable products (Fekairi et al., 2009).In addition to the mechanisms described above, an activity resembling that of EcRuvC had long been known to be present in mammalian cell-free extracts. In 2008, the group of Steven C. West succeeded in identifying, to their knowledge, the first nuclear proteins analogous to the EcRuvC paradigm: ScYen1 and Homo sapiens XPG-like endonuclease1 (HsGEN1; Ip et al., 2008). These proteins are members of the large and well-characterized Rad2/XPG family of nucleases. The Rad2/XPG family consists of the Xeroderma pigmentosum group G-complementing protein (XPG) endonucleases of the nucleotide excision repair (class I), the flap endonuclease1 (FEN1) replication-associated flap endonucleases (class II), the exodeoxyribonuclease1 (EXO1) exonucleases of recombination and repair (class III), and class IV (containing the [putative] eukaryotic HJ resolvases). This last class was introduced after the identification of the rice (Oryza sativa) single-strand DNA endonuclease1 (OsSEND-1) based on sequence homology. The class IV members show a domain composition homologous to FEN1 and EXO1, with no spacer region between their N-terminal XPG (XPG-N) and internal XPG (XPG-I) domains, whereas the primary structure of these domains is more similar to the sequence of the nuclease domain of XPG (Furukawa et al., 2003).Although all Rad2/XPG homologs share a common cleavage mechanism as observed for the typical 5′-flap substrate (Tsutakawa et al., 2011; Tsutakawa and Tainer, 2012), the striking evolutionary difference between classes I, II, and III on the one hand and the HJ resolvases (class IV) on the other hand is the ability of class IV members to form homodimers in vitro at their preferred substrate, the HJs (Rass et al., 2010). The homodimer configuration ensures the presence of two active sites positioned on the opposing strands of the HJ, which is necessary for resolution. The mode of eukaryotic HJ resolution is largely similar to the bacterial paradigm: (1) cleavage occurs one nucleotide in the 3′ direction of a static junction point (equivalent to the main cleavage site on 5′-flaps), (2) the incisions occur with almost perfect point symmetry, (3) the incisions result in readily ligatable nDs, and (4) certain sites within a migratable HJ core are preferred, providing evidence for a (yet to be determined) sequence specificity (Ip et al., 2008; Bailly et al., 2010; Rass et al., 2010; Yang et al., 2012).In the absence of MUS81-EME1/Mms4, the proteins HsGEN1, ScYen1, and CeGEN-1 have been shown to play a role in response to replication-associated perturbations, such as MMS- and UV-induced DNA damage (Bailly et al., 2010; Blanco et al., 2010; Tay and Wu, 2010; Gao et al., 2012; Muñoz-Galván et al., 2012). It is also likely that these proteins provide a backup mechanism in mitosis and meiosis, ensuring proper chromosome segregation after a failure of other mechanisms, including MUS81-EME1/Mms4 (Blanco et al., 2010; Matos et al., 2011).Although canonical HJ resolvases in animals and fungi are a current topic of great interest, very little is known about these proteins in plants. In rice, two members of the Rad2/XPG class IV have been described: OsSEND-1 (the founding member) and OsGEN-like (OsGEN-L). OsSEND-1 was shown to digest single-stranded circular DNA, and its expression is induced on MMS-induced genotoxic stress, whereas OsGEN-L is implicated in late spore development (Furukawa et al., 2003; Moritoh et al., 2005). Both studies (Furukawa et al., 2003; Moritoh et al., 2005) proposed putative homologs in other plants, and the gene locus At1g01880 of Arabidopsis, coding for the protein AtGEN1, is considered the ortholog of HsGEN1 and ScYen1 (Ip et al., 2008). However, currently, only OsGEN-L has been further investigated and described to possess in vitro properties similar to both Rad2/XPG nucleases and EcRuvC. This protein shows a well-defined 5′-flap activity as well as a poorly characterized ability, similar to that of EcRuvC, to resolve mobile HJs (Yang et al., 2012).Thus, of two members of Rad2/XPG class IV of plants, only one member has so far been analyzed with respect to a possible HJ resolvase activity. However, Arabidopsis expression data show that both proteins are expressed in plants and do not reveal marked differences (Laubinger et al., 2008). In this study, the goal was, therefore, to characterize the in vitro activities of not only AtGEN1 but also, AtSEND1, focusing on the idea that Arabidopsis and (seed) plants in general might encode not one but actually two HJ resolvases with functional homology to EcRuvC.