A Holliday junction resolvase from Pyrococcus furiosus: functional similarity to Escherichia coli RuvC provides evidence for conserved mechanism of homologous recombination in Bacteria, Eukarya, and Archaea

Abstract

The Holliday junction is an essential intermediate of homologous recombination. RecA of Bacteria, Rad51 of Eukarya, and RadA of Archaea are structural and functional homologs. These proteins play a pivotal role in the formation of Holliday junctions from two homologous DNA duplexes. RuvC is a specific endonuclease that resolves Holliday junctions in Bacteria. A Holliday junction-resolving activity has been found in both yeast and mammalian cells. To examine whether the paradigm of homologous recombination apply to Archaea, we assayed and found the activity to resolve a synthetic Holliday junction in crude extract of Pyrococcus furiosus cells. The gene, hjc (Holliday junction cleavage), encodes a protein composed of 123 amino acids, whose sequence is not similar to that of any proteins with known function. However, all four archaea, whose total genome sequences have been published, have the homologous genes. The purified Hjc protein cleaved the recombination intermediates formed by RecA in vitro. These results support the notion that the formation and resolution of Holliday junction is the common mechanism of homologous recombination in the three domains of life.

Figure 1

Partial purification of Holliday junction cleavage activity from P. furiosus. (A) SDS/PAGE (15%) of the fractions separated by heparin-Sepharose column. All bands in the gel were visualized by silver staining. Protein bands indicated by arrows were eluted in correspondence with the activity. (B) Holliday junction cleavage activity was found in the fractions from the heparin-Sepharose column chromatography. Aliquots of the column fractions (fractions 19–32), together with buffer A (B) and peak fraction from hydroxyapatite chromatography, Fraction IV (IV), were incubated with ³²P-labeled 4Jh. The products were analyzed by 12% PAGE followed by autoradiography.

Figure 2

Cloning and expression of the gene responsible for Holliday junction cleavage activity. (A) Restriction map around the gene encoding Holliday junction cleaving enzyme and the results of deletion mutant analysis. B, BamHI; E, EcoRI; Ev, EcoRV; E22, EcoT22I; H, HindIII; Nc, NcoI; Sa, SacI; Sc. ScaI; Sl. SalI; Xb, XbaI; Xh, XhoI. The ORFs found from the nucleotide sequence are indicated by arrows. (B) Result of the Holliday junction cleavage assay by using the heat-treated supernatants derived from the cell extract of E. coli clones that have plasmids carrying each restriction fragment (–8) indicated in A. The crude extract of P. furiosus was used as a control (P.fu). S and P indicate the substrate and product bands, respectively. (C) SDS/PAGE (15%) analysis of the recombinant Hjc protein from the different stages of the purification. Lanes: 1, supernatant after sonication; 2, polyethylenimine fraction; 3, ammonium sulfate fraction; 4, supernatant after heat treatment; 5, hydroxyapatite fraction; 6, Mono Q fraction. Lane M contains size marker proteins (Bio-Rad). The gel was stained with Coomassie brilliant blue. Arrowhead indicates the Hjc band.

Figure 3

Immunological identification of Hjc in P. furiosus cells. (A) The sonication extract of recombinant cells was separated by 15% SDS/PAGE, transferred onto PVDF membranes and treated with immune (I) or preimmune (P) sera raised against Hjc. (B) Fraction V from P. furiosus cells (Pfu) and 17 ng of recombinant Hjc protein (R) were analyzed by Western blotting with anti-Hjc serum. (C) Immunodepletion analysis. (Top) Endonuclease assay of each depleted extract. (Bottom) Each precipitant was analyzed by Western blotting with anti-Hjc serum. Lanes: B, buffer A; 1, crude extract before antiserum treatment; 2, anti-Hjc serum; 3, anti-Pfu-PCNA serum; 4, anti-PI-PfuI serum. Lane R was loaded with 15 ng of recombinant Hjc.

Figure 4

Determination of the cleavage sites produced by the Hjc. An immobile four-way junction (A and B) and a mobile junction (C and D), each labeled uniquely at the 5′-³²P-end in indicated (∗) strand, were used as substrates. Substrate DNAs were incubated with (+) or without (−) Hjc protein. The GA sequence ladders of each labeled oligonucleotide generated by the Maxam–Gilbert method were loaded alongside to provide markers. The cleavage sites are represented by arrowheads showing cutting efficiencies by their sizes. The box part of the junctions in D indicates the mobile homologous region.

Figure 5

Cleavage of the RecA-mediated recombination intermediate by Hjc. (A) Scheme of the formation of the Holliday junctions by RecA and the resolution of the junctions by resolvase. Asterisks show the 3′-³²P-labeled ends. (B) Recombination intermediates between gapped DNA and homologous ³²P-labeled linear duplex DNA were formed by RecA and then were treated by indicated concentration of Hjc (lanes 5–11) or Fraction V from P. furiosus (lane 12) at 55°C for 10 min or by 0.17 μM of RuvC (lane 13) at 37°C for 1 hr. The RecA-mediated products that are from incubation of the substrates with RecA for 30 min at 37°C (lane 3) and the intermediates before Hjc treatment (lane 4) were loaded as controls. ³²P-labeled linear duplex DNA (3 ng) was loaded as a marker (lane 1). Recombination products were analyzed by 1.2% agarose gel electrophoresis.

Figure 6

Comparison of Hjc amino acid sequence with homologs found in four archaeal strains (A) and E. coli RuvC (B). Pfu, P. furiosus; Pho, P. horikoshii; Mja, M. jannaschii; Afu, A. fulgidus; Mth, M. thermoautotrophicum. Identical and similar amino acid residues are indicated by red and blue letters, respectively. The four residues indicated by green letters in RuvC are known to constitute the catalytic center (42, 8).