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Drosophila melanogaster XPG-like endonuclease (DmGEN) is a new category of nuclease belonging to the RAD2/XPG family. The DmGEN protein has two nuclease domains (N and I domains) similar to XPG/class I nucleases; however, unlike class I nucleases, in DmGEN these two nuclease domains are positioned close to each other as in FEN-1/class II and EXO-1/class III nucleases. To confirm the properties of DmGEN, we characterized the active-site mutant protein (E143A E145A) and found that DmGEN had flap endonuclease activity. DmGEN possessed weak nick-dependent 5′−3′ exonuclease activity. Unlike XPG, DmGEN could not incise the bubble structure. Interestingly, based on characterization of flap endonuclease activity, DmGEN preferred the blocked-flap structure as a substrate. This feature is distinctly different from FEN-1. Furthermore, DmGEN cleaved the lagging strand of the model replication fork. Immunostaining revealed that DmGEN was present in the nucleus of actively proliferating Drosophila embryos. Thus, our studies revealed that DmGEN belongs to a new class (class IV) of the RAD2/XPG nuclease family. The biochemical properties of DmGEN and its possible role are also discussed.
DNA replication, recombination and repair are key processes in maintaining genome integrity. Nucleases are necessary for their nucleolytic activities. They act on a variety of structural frameworks, ranging from site-specific (e.g. AP endonuclease) to structure-specific (e.g. RAD2/XPG nuclease family) and nonspecific (e.g. DNase I) nucleases. In particular, members of the RAD2/XPG nuclease family have unique nuclease activities and play critical roles in genome stability [1–6]. In a preliminary report, we described the presence of a new nuclease, Drosophila melanogaster XPG-like endonuclease (DmGEN) which belongs to the RAD2/XPG nuclease family, shows unique activity and possibly plays a critical role in genome stability . The ORF of the DmGEN gene encoded a predicted protein of 726 amino acid residues with a molecular mass of 82.5 kDa. The gene was located at 64C9 on the left arm of Drosophila polytene chromosome 3 as a single site.
The RAD2/XPG family of nucleases, which have two conserved nuclease domains (the N domain and the I domain), are currently separated into three classes (XPG/class I, FEN-1/class II and EXO-1/class III) based on the types of nuclease activity and sequence homology [8,9]. In Drosophila, mus201 protein (class I), FEN-1 homologue protein (class II), and Tosca protein (class III) have been reported as RAD2 family proteins. The DmGEN protein showed a relatively high degree of sequence homology with RAD2 nucleases, particularly XPG, although the locations of the N and I domains were similar to those of FEN-1 and EXO-1, and the molecular mass of DmGEN was found to be close to that of EXO-1. Therefore, we proposed a new class (class IV) to categorize DmGEN and SEND-1, which we also found in higher plants . Recently, a new member of the class IV nucleases, OsGEN-like, has been reported in rice; RNA-mediated silencing of the OsGEN-like caused male sterility due to a defect in microspore development . Although DmGEN homologues are found widely in mammals and higher plants [7,9], knowledge about their biochemical properties is limited. In this study, we determined the biochemical properties of native and an active-site mutant DmGEN to more deeply understand the nature of this new class of nucleases.
As for the biochemical features, class I consists of XPG homologues, which cleave at the 3′ side of the bubble structure formed during nucleotide excision repair [10,11]. Class II comprises the FEN-1 homologues, which show 5′-flap endonuclease, 5′−3′ exonuclease and gap endonuclease activities, and play important roles in RNA primer removal, base excision repair and apoptotic DNA fragmentation [12–14]. Class III is made up of the EXO-1 homologues, which have 5′−3′ exonuclease activity and are involved in DNA recombination, mismatch repair and DNA replication [15–18]. The function for class IV, however, remains unclear. In relation to the studies, we must correct some mistakes in our previous study. We reported previously that DmGEN has not only 3′−5′- and nick- and gap-dependent 5′-3′ exonuclease activities, but also endonuclease activity at a site 3 or 4 bp from the 5′-end . However, such activities were not found when DmGEN was purified more carefully, although nick-dependent 5′-3′ exonuclease activity was present. Thus, it is important to re-characterize this novel enzyme.
Here, we report that the DmGEN protein is a new flap endonuclease, which is different in nature from FEN-1. Based on our studies we have confirmed that DmGEN belongs to a new class of the RAD2/XPG family. In addition, we have characterized the biochemical properties of DmGEN.
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The purpose of this study was to precisely characterize a newly found member (class IV) of the RAD2/XPG family of nucleases, DmGEN, from Drosophila melanogaster. The biochemical properties of class IV nucleases are largely unknown in various animals and plants. For this purpose, we created an active-site mutant, and used this mutant to confirm the biochemical properties of DmGEN. We purified wild-type and mutant DmGEN protein using an improved purification protocol, and analyzed the nuclease activities of the purified proteins. Thus, we showed that DmGEN was a new type of flap endonuclease.
The amino acid sequence of DmGEN protein has three principal features (Fig. 2). First, one of the acidic residues at the active center for catalysis was not conserved in DmGEN (Fig. 2B, asterisk 1). Regarding this nonconserved aspartic acid residue, Constantinou et al.  reported that the D77E active-site mutant of XPG protein showed considerably lower nuclease activity than wild-type XPG protein. Second, most of the positively charged amino acids residues, which are essential for binding FEN-1 to DNA [14,20], were not conserved in DmGEN protein (Fig. 2C, Table 2). These two features contribute to the low nuclease activity of DmGEN. Lastly, DmGEN shows high homology between the N and I regions and XPG (class I), but the spacing of these regions is similar to in FEN-1 and EXO-1 (class I and III, respectively). We confirmed how this feature contributes the nuclease activity of DmGEN (class IV). DmGEN had a flap endonuclease activity, like FEN-1, but was not able to cleave the bubble structure, unlike XPG (Figs 3, 4). Because DmGEN had no 5′−3′ exonuclease activity on the dsDNA substrate (Fig. 3D), DmGEN is distinctly different from EXO-1 (class III). Recently, it was suggested that the activity of the RAD2/XPG nuclease family is determined by the properties and positions of the two nuclease domains [19,28]. The adjacent position of the two domains may be responsible for not cleaving the bubble structure (Fig. 4C), because a XPG mutant with a deletion in the spacer region was shown to prefer the pseudo Y structure to the bubble structure .
The flap endonuclease activity of DmGEN is more accurate and weaker than that of FEN-1 (class II nuclease). For example, FEN-1 cleaves many DNA structures such as 5′-single-strand overhang including flap, pseudo Y, gapped-flap and 5′-overhang double-strand . In contrast, as shown Fig. 6, DmGEN cleaves the normal flap substrate and a special flap structure: the blocked-flap substrate in which the 5′-single-strand overhang of the flap is double-stranded; DmGEN cleaves just at the ssDNA/dsDNA junction point. We found very little cleavage of gapped-flap and pseudo Y substrates by DmGEN. Therefore, the DNA structure at the junction seems to be important for DmGEN-mediated cleavage. Unlike pseudo Y and gapped-flap, DmGEN preferred a substrate in which the 5′-upstream of the flap is completely double-stranded. This idea is supported by the fact that DmGEN cleaved the double-flap substrate (Fig. 6). The interesting feature of DmGEN is that this nuclease cleaves the blocked-flap structure, and this activity is slightly stronger than the normal flap structure cleaving activity, a feature that is distinctly different from that of FEN-1. In agreement with the previous report , we also found that the activity of FEN-1 decreases considerably when the flap substrate is double-stranded (Fig. 7A). On the hairpinned-flap substrates having no free 5′-end, the nuclease activity of both FEN-1 and DmGEN are weaker than that on the normal flap substrate (Fig. S1). Because FEN-1 prefers a free 5′ ssDNA end of flap [14,23,24], the nuclease activity on the hairpinned-flap substrate is weak, like for the blocked-flap substrate . Therefore, in contrast to FEN-1, DmGEN prefers a free 5′-end of flap, which is either single- or double-stranded, this is deduced from the fact that DmGEN preferred the blocked-flap structure, but not the hairpinned-flap structure. These results suggest that binding of the substrate to DmGEN might differ from that of FEN-1. This is also suggested by the fact that most of the positively charged amino acids residues, which are essential for binding of FEN-1 to DNA , were not conserved in the DmGEN protein (Fig. 2C).
The most interesting activity of DmGEN is that it cleaves the blocked-flap structure and the hairpinned-flap structure substrate (Fig. 7B, Fig. S1). The blocked-flap structure can be regarded as a model for the normal replication fork. Interestingly, DmGEN cleaved the lagging strand of the model replication fork with gaps (Fig. 7C). Furthermore, DmGEN was localized in the nucleus of Drosophila early embryos, in which DNA replication is actively occurred (Fig. 8). To maintain chromosome integrity, several DNA repair pathways coupled with the lagging strand of the replication fork are working [29,30]. Lopes et al. reported that ssDNA gaps accumulate along replicated duplexes in vivo, when DNA replication forks pause and restart near lesions on the template . They also argued that translesion synthesis and recombinational repair play a crucial role in repairing these ssDNA gaps . RAD51-mediated DNA recombinational repair needs free 3′-overhang DNA [32,33]. We speculate that DmGEN may cleave the lagging strand of the replication fork with gaps. As a result of the cleavage, DmGEN produces free 3′-overhang DNA, which subsequently becomes available for RAD51-mediated recombinational repair.
The above-described properties of DmGEN obviously differ from other members of the RAD2/XPG family of nucleases. Thus, we suggest that DmGEN should be categorized in a new class (class IV) of the RAD2/XPG family. Homologues of DmGEN are widely found in animals and plants. This suggests that DmGEN may play an important biological role. Further characterization of DmGEN may shed new light on biological events related to DNA metabolism.