Distinct activities of exonuclease 1 and flap endonuclease 1 at telomeric g4 DNA

Abstract

Background: Exonuclease 1 (EXO1) and Flap endonuclease 1 (FEN1) are members of the RAD2 family of structure-specific nucleases. Genetic analysis has identified roles for EXO1 and FEN1 in replication, recombination, DNA repair and maintenance of telomeres. Telomeres are composed of G-rich repeats that readily form G4 DNA. We recently showed that human EXO1 and FEN1 exhibit distinct activities on G4 DNA substrates representative of intermediates in immunoglobulin class switch recombination.

Methodology/principal findings: We have now compared activities of these enzymes on telomeric substrates bearing G4 DNA, identifying non-overlapping functions that provide mechanistic insight into the distinct telomeric phenotypes caused by their deficiencies. We show that hFEN1 but not hEXO1 cleaves substrates bearing telomeric G4 DNA 5'-flaps, consistent with the requirement for FEN1 in telomeric lagging strand replication. Both hEXO1 and hFEN1 are active on substrates bearing telomeric G4 DNA tails, resembling uncapped telomeres. Notably, hEXO1 but not hFEN1 is active on transcribed telomeric G-loops.

Conclusion/significance: Our results suggest that EXO1 may act at transcription-induced telomeric structures to promote telomere recombination while FEN1 has a dominant role in lagging strand replication at telomeres. Both enzymes can create ssDNA at uncapped telomere ends thereby contributing to recombination.

Figure 1. hFEN1, but not hEXO1, cleaves 5′ telomeric G4 DNA.

(A) Diagram of substrates bearing a G4 or unstructured 5′ flaps. Lengths of oligonucleotides, flaps and duplex regions are indicated; asterisks denotes 5′ end-label. (B) Products of digestion of 5′ flap substrates shown in Panel A by hEXO1 (0, 1.2, 2.4 and 3.6 nM) and hFEN1 (0, 4.5, 9.0 and 18 nM). Arrows indicate undigested (60 nt or 36 nt) 5′-labeled DNA substrate, products of flap endonuclease digestion, and 1 nt product of exonuclease digestion. Heterogenous flap cleavage products like those evident here are characteristic of hFEN1 activity . (C) Quantitation of flap cleavage activity of hEXO1 (0, 1.2, 2.4 and 3.6 nM; diamonds) and hFEN1 (0, 4.5, 9 and 18 nM; squares) on substrates shown in Panel A.

Figure 2. A 3′ G4 DNA tail inhibits flap endonuclease activity of hEXO1 but not hFEN1.

(A) Diagram of substrates bearing a 5′ flap and 3′ telomeric G4 DNA or polyA tails. Lengths of oligonucleotides, flaps, duplex regions and 3′ tails are indicated; asterisks denote 5′ end-label. (B) Products of digestion of substrates shown in Panel A by hEXO1 (0, 1.2, 2.4 and 3.6 nM) and hFEN1 (0, 4.5, 9.0 and 18 nM). Arrows indicate 60 nt 5′-labeled DNA substrate, 16 nt flap cleavage product, and 1 nt exonucleolytic cleavage product. (C) Quantitation of flap cleavage activity of hEXO1 (0, 0.6, 1.2 and 2.4 nM; diamonds) and hFEN1 (0, 4.5, 9 and 18 nM; squares) on substrates shown in Panel A.

Figure 3. hFEN1, but not hEXO1, cleaves 5′ nontelomeric G4 DNA flaps and 5′ flaps adjacent to 3′ nontelomeric G4 DNA tails.

(A) Diagram of substrates bearing 5′ or 3′ nontelomeric G4 DNA tails. Lengths of oligonucleotides, flaps, duplex regions and 3′ tails are indicated; asterisk denotes 5′ end-label. (B) Products of digestion of substrates shown in Panel A by hEXO1 (0, 1.2, 2.4 and 3.6 nM) and hFEN1 (4.5, 9.0 and 18.0 nM). Arrows indicate 60 nt undigested 5′-labeled DNA substrates, products of flap endonuclease digestion, and 1 nt product of exonuclease digestion. (C) Quantitation of flap cleavage activity of hEXO1 (0, 0.6, 1.2 and 2.4 nM; diamonds) and hFEN1 (0, 4.5, and 9.0 nM; squares) on substrates shown in Panel A.

Figure 4. hEXO1 and hFEN1 excise from a nick on a strand bearing a 3′ telomeric tail.

(A) Diagram of duplex substrates bearing an internal nick adjacent to a 5′ end-labeled 3′-G4 DNA (TTAGGG)₄ tail, 3′ poly(A)₂₄ tail, or blunt end. The length of the labeled oligonucleotide is indicated; asterisks, end-label. (B) Products of digestion of each substrate by hEXO1 (0, 0.6, 1.2 and 2.4 nM). Arrows indicate 42 or 18 nt undigested 5′-lableled DNA substrate and 1 nt excision product. (C) Quantitation of hEXO1 excision of blunt-ended duplex substrates (squares), substrates bearing 3′ G4 DNA overhangs (diamonds) and substrates bearing poly(A)₂₄ tails (triangles). (D) Products of digestion by hFEN1 (0, 4.5, 9.0 and 18 nM). Notations as in panel B. (E) Quantitation of hFEN1 excision of blunt-ended duplex substrates (squares), substrates bearing 3′ G4 DNA overhangs (diamonds) and substrates bearing polyA tails (triangles).

Figure 5. A telomeric tail stimulates hEXO1 and hFEN1 excision on the opposite strand.

(A) Diagram of duplex substrates for assay of excision opposite a G4 DNA (TTAGGG)₄ tail, a polyA₂₄ tail, or a blunt end. Sizes of 3′-labeled oligonucleotides are indicated; asterisks, end-label. (B) Quantitation of hEXO1 (0, 0.6, 1.2 and 2.4 nM, above) or hFEN1 (0, 4.5, 9.0 and 18 nM, below) excision of substrates diagrammed in panel A. G4 DNA tails, triangles; 3′ poly(A)₂₄ tails, squares and blunt ends, circles. (C) Diagram of duplex substrates for assay of excision at a nick on the strand opposite a G4 DNA (TTAGGG)₄ tail, a 3′-poly(A)₂₄ tail, or a blunt end, and 3′ end-labeled on the nicked strand distal to the tail. Sizes of 3′-labeled oligonucleotides are indicated; asterisks, end-label. (D) Quantitation of hEXO1 (0, 0.6, 1.2 and 2.4 nM, above) or hFEN1 (0, 4.5, 9.0 and 18 nM, below) excision of substrates diagrammed in panel C. Notations as in panel B.

Figure 6. Transcribed telomere repeats are excised by hEXO1 but not hFEN1.

(A) pTELN telomeric substrates. T7 promoter (P_T7); 800 human (TTAGGG) telomeric repeat (dark fill); RNA transcript (dashed line); and PvuII cleavage sites (Pv; arrows) are indicated. (B) Products of PvuII digests of pTELN substrates, which had been transcribed or nicked, as indicated, following digestion with hEXO1 (above, 0.6, 1.2, 2.4 and 4.8 nM) or hFEN1 (below, 0. 4.5, 9 and 18 nM). Fragments were phosphor-imaged and quantitated following transfer and indirect labeling with a probe to the T7 promoter. Arrows denote the 3.8 kb full-length plasmid, and the 2.5 kb PvuII product. (C) Quantitation of products of excision of transcribed, nicked pTELN (diamonds), transcribed, pTELN (squares), nicked pTELN (triangles) and supercoiled pTELN (open circles) by 0, 1.2, 2.4 and 3.6 nM hEXO1 (left) and by 0, 4.5, 9 and 18 nM hFEN1 (right). Open circles and squares are overlapping for hEXO1.