AtGEN1 and AtSEND1, two paralogs in Arabidopsis, possess holliday junction resolvase activity

Abstract

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.

Figure 1.

Eukaryotic HJ resolvases. A, Domain structure of subclass IV of the Rad2/XPG superfamily. All members share a common N-terminal domain organization, whereas the C-terminal half contains no defined functional domains. XPG-N and XPG-I domains constitute the nuclease domain and mediate metal ion coordination by conserved amino acids. The helix-hairpin-helix2 domain is implicated in binding of double-stranded DNA. B, The evolutionary history was inferred using the maximum parsimony method. Tree 1 of the two most parsimonious trees (length = 2,351) is shown. AtMUS81 was defined as the outgroup. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The maximum parsimony tree was obtained using the Subtree-Pruning-Regrafting algorithm with search level 1, in which the initial trees were obtained by the random addition of sequences (10 replicates). aa, Amino acid.

Figure 2.

Structure-specific cleavage of flapped DNA structures. A, Incision mapping at four static 5′-flaps (1–4; indicated by the respective colors) with different sequence compositions. The dots mark the labeled 5′ end of the cleaved flapped strand. The length of the arrowheads and percentages give the 31-nucleotide product as the portion of the total DNA content. B, AtGEN1 and AtSEND1 cleave the four 5′-flaps with different efficiencies: AtGEN1 is less active with 5′-flap3, and AtSEND1 is less active with 5′-flap2 (native gel electrophoresis). C, Sequence context of the flapped strands around the junction point. The main and secondary cleavage sites of AtGEN1 and AtSEND1 are indicated by arrows. D, AtGEN1 and AtSEND1 do not cleave 3′-flaps as shown by denaturing gel electrophoresis of the 5′-labeled flapped strand. E, Cleavage of a RF (RF2) by AtGEN1 and AtSEND1 occurs exclusively in the matrix strand of lagging strand synthesis and preferentially around the branch point of the junction. The cleavage positions, which are equivalent to the lengths of the cleaved oligonucleotides measured from the 5′ end, are indicated at or in the arrowheads. The dotted lines demarcate the migratable homologous core of the RF. nt, Nucleotide.

Figure 3.

Schematic summary of incision mapping at the static HJ X0 with AtGEN1 (A) and AtSEND1 (B). Cleavage events (Supplemental Fig. S4) were quantified, and the relative frequencies are represented by the lengths of the arrows. The cleavage positions, which are equivalent to the lengths of the cleaved oligonucleotide measured from the 5′ end, are indicated at or in the arrowheads. Cleavage products that represent less than 5% of the cleavage events are not shown.

Figure 4.

Symmetric cleavage pattern at a HJ providing a migratable, homologous core (HJ X26). Four versions of the X26 were used as substrates (differing in which strand was labeled; numbers with asterisks). A, Native gel electrophoresis of the reaction products of AtGEN1 and AtSEND1 compared with buffer controls (−). With the help of marker structures, the reaction products were identified. B, Quantification of the results of independent experiments as shown in A. C and D, Schematic summary of incision mapping at the mobile HJ X26 with AtGEN1 (C) and AtSEND1 (D). The cleavage events (Supplemental Fig. S5) were quantified, and the relative frequencies are represented by the lengths of the arrows. The cleavage positions, which are equivalent to the lengths of the cleaved oligonucleotide measured from the 5′ end, are indicated at the arrowheads. Cleavage products that represent less than 5% of the cleavage events are not shown. The lengths of the main products are indicated.

Figure 5.

Ligation of cleavage products. A, Schematic illustration of the ligation assay principle. The asymmetric X26-S HJ containing a label (red dot on red oligonucleotide) was used. Symmetric cleavage followed by ligation yields a longer labeled oligonucleotide of 60 nucleotides (red and yellow segments). B, Resolution products of AtGEN1 and AtSEND1 are ligatable, which is shown by a denaturing sequencing gel after performing the experiments according to the scheme in A. nt, Nucleotide.

Figure 6.

Resolvase and nicking activities of AtGEN1 and AtSEND1 on plasmid pIR9. A, The plasmid pIR9 features an inverted repeat, which forms a HJ stabilized by negative supercoiling. A single incision leads to relaxation and reabsorption of the HJ if the junction is not stabilized by the DNA-protein complex. The resulting nicked circular (n.c.) plasmid, therefore, is a nicking product. Resolution into linear (lin.) products through two opposing incisions can happen either simultaneously or in a successive manner in which the HJ structure is maintained by the bound resolvase dimer. B, Separation of the nicking and resolution products from the uncleaved supercoiled (sc.) plasmid. For quantification, the percentage of the products was corrected for the background of nicked circular and linear DNA in the buffer reaction (−). The fraction of supercoiled plasmid resistant to EcoRI digestion was determined and set to 100%, representing the available portion of plasmids extruding the cruciform structure.

Figure 7.

nHJs are primarily cut in the strand opposite the nick but also in the strand that hybridizes with the 5′ end of the nick. Results of incision mapping of two different nHJs treated with AtGEN1 or AtSEND1 are shown. A, AtGEN1 at nX-30. B, AtSEND1 at nX-30. C, AtGEN1 at nX-32. D, AtSEND1 at nX-32. The cleavage events were quantified, and the relative frequencies are represented by the lengths of the arrows. Cleavage products that represent less than 5% of the cleavage events are not shown. The cleavage positions equivalent to the lengths of the main products are indicated (in nucleotides) in or at the arrowheads.

Figure 8.

nHJs are processed through both a resolution-like pathway and a pathway involving a RF intermediate (Ref-I pathway). In the resolution-like pathway, the position of incision in the strand opposing the nick is dependent on the sequence context at the junction point. A, The nHJ nX-30 was labeled on strand 1, and native gel electrophoresis after incubation with AtGEN1 or AtSEND1 reveals more products than the nD structure expected by a solely canonical HJ resolvase. The sD1* product is the result of a first incision in strand 4, yielding a RF intermediate, and then a second in strand 1 (see text). The nD1* and sD1* products were analyzed separately on sequencing gels to identify the exact cleavage positions for the two different pathways. Supporting data regarding the identity and processing of the RF intermediates are presented in Supplemental Figures S7 and S8. B, Assignment of the cleavage events for the nX-30 to the two different pathways. The portions of the arrowheads ascribed to resolvase-like (dark colors) and Ref-I (light colors) pathways are based on the quantifications shown in Figure 7 and Supplemental Figure S8, H to K. The cleavage positions, which are equivalent to the lengths of the 5′ cleavage products, are indicated in or at the colored arrowheads. The relative frequencies are represented by the lengths of the arrows. Cleavage products that represent less than 5% of the cleavage events are not shown. C is like A but with the nX-32. D, Assignment of the cleavage events for the nX-32 to the two different pathways, like in B.

Figure 9.

Summary of tested substrate types and detected activities. All types of oligonucleotide-based substrates used in the in vitro experiments are shown, and the corresponding activities of AtGEN1 (violet) and AtSEND1 (green) are presented in a simplified manner. The length of the arrows is a rough approximation of the activities of the enzymes on all tested substrate subtypes (e.g. RF1–RF3) to present a more general picture than shown in the detailed data presentation before. The schematic of the mobile nHJs is to be read as follows: AtGEN1 cleaves strand 1 of the nX-32 exactly opposite the discontinuity in strand 3 (continuous arrows), leading to religatable products, whereas for the nX-30, the inherent preference of AtGEN1 to cleave at a certain site in relation to the junction point leads to an incision one nucleotide 3′ of the junction point (dashed arrows). For AtSEND1, the situation is inverted. Cleavage of strand 4 of the nHJs (small arrows) leads to a RF intermediate, which is processed as shown for the RF substrate type. Note that the Ref-I activity can also be observed at the static X0 and to a limited degree, the intact X26. nt, Nucleotide.