Methanococcus jannaschii flap endonuclease: expression, purification, and substrate requirements

Abstract

The flap endonuclease (FEN) of the hyperthermophilic archaeon Methanococcus jannaschii was expressed in Escherichia coli and purified to homogeneity. FEN retained activity after preincubation at 95 degrees C+ for 15 min. A pseudo-Y-shaped substrate was formed by hybridization of two partially complementary oligonucleotides. FEN cleaved the strand with the free 5' end adjacent to the single-strand-duplex junction. Deletion of the free 3' end prevented cleavage. Hybridization of a complementary oligonucleotide to the free 3' end moved the cleavage site by 1 to 2 nucleotides. Hybridization of excess complementary oligonucleotide to the free 5' end failed to block cleavage, although this substrate was refractory to cleavage by the 5'-3' exonuclease activity of Taq DNA polymerase. For verification, the free 5' end was replaced by an internally labeled hairpin structure. This structure was a substrate for FEN but became a substrate for Taq DNA polymerase only after exonucleolytic cleavage had destabilized the hairpin. A circular duplex substrate with a 5' single-stranded branch was formed by primer extension of a partially complementary oligonucleotide on virion phiX174. This denaturation-resistant substrate was used to examine the effects of temperature and solution properties, such as pH, salt, and divalent ion concentration on the turnover number of the enzyme.

FIG. 1

SDS-PAGE analysis of M. jannaschii FEN protein on a HiTrap SP column. Lanes: 1, molecular weight markers; 2, fraction after heating at 70°C; 3 to 8, flowthrough and low-salt wash fractions; 9 and 10, 0.3 M NaCl elution fractions.

FIG. 2

Diagrams of FEN substrates. (A) Pseudo-Y structure formed by annealing oligonucleotides FS and FA; (B) structure formed by annealing a primer (P-4, P-6, or P-15) to φX174 virion DNA and extending; (C) substitution of oligonucleotide FA-T for FA; (D) annealing of oligonucleotide FA-C to FA; (E) annealing of oligonucleotide FS-C to FS; (F) ligation of oligonucleotide FS-L to FS.

FIG. 3

Cleavage of pseudo-Y structure at 50°C. Lanes: 1 to 3, size markers (10, 15, and 21 nt, respectively); 4, oligonucleotide FS (37 nt) with truncated sequences; 5, FS after incubation of FS plus FA with Taq DNA polymerase; 6, FS after incubation of FS plus FA with FEN; 7, FS after incubation of FS alone with Taq DNA polymerase.

FIG. 4

Thermostability of FEN protein. Lanes: 1, 21-nt marker; 2, oligonucleotide FS (37 nt); 3, FS after incubation of FS plus FA with FEN; 5, FS after incubation of FS plus FA with unheated FEN protein; 7, FS after incubation of FS plus FA with FEN protein preheated to 90°C for 15 min; lanes 4, 6, and 8, same as lanes 3, 5, and 7 but with omission of oligonucleotide FA.

FIG. 5

Time course of cleavage of a pseudo-Y structure at 50°C. Lanes 1 to 4 represent reactions stopped at 16, 32, 45, and 90 min, respectively.

FIG. 6

Removal of a flap at a restriction endonuclease cleavage site at 55°C. Lanes: 1 to 6, φX174 primer extension product cut with BstXI and BsiEI; 1, no preincubation with FEN protein; 2 to 6, preincubation with a twofold serial dilution of FEN protein, with 50 pg (in 20 μl) in lane 6; 7, markers.

FIG. 7

Dependence of FEN activity on temperature (A) and pH (B). All reaction mixtures contained 2 pmol of FEN protein in 50 μl.

FIG. 8

Activity of FEN on modified pseudo-Y substrates at 50°C. (A) Lanes: 1, marker (FS, 37 nt); 2 and 3, FS after incubation of FS plus FA with Taq DNA polymerase and FEN, respectively; 4 and 5, FS after incubation of FS plus FA-T with Taq DNA polymerase and FEN, respectively (see Fig. 2C); 6 and 7, FS after incubation of FS, FA, and FA-C (hybridized to FA) with Taq DNA polymerase and FEN, respectively (see Fig. 2D). (B) Lanes: 1, 21-nt marker; 2, oligonucleotide FS (37 nt); 3 and 5, FS after incubation of FS plus FA (unmodified substrate) with Taq DNA polymerase and FEN, respectively; 4 and 6, FS after incubation of FS, FA, and FS-C (hybridized to FS) with Taq DNA polymerase and FEN, respectively (see Fig. 2E). (C) Lanes: 1, marker formed by ligating oligonucleotide FS-L to ³²P-labeled FS to form a hairpin loop (see Fig. 2F); 2 to 4, hairpin loop after incubation with Taq DNA polymerase for 5, 15, and 45 min, respectively; 5 to 7, hairpin loop after incubation with FEN for 5, 15, and 45 min, respectively.