Human exonuclease 1 (EXO1) activity characterization and its function on flap structures

Abstract

Human exonuclease 1 (EXO1) is involved in multiple DNA metabolism processes, including DNA repair and replication. Most of the fundamental roles of EXO1 have been described in yeast. Here, we report a biochemical characterization of human full-length EXO1. Prior to assay EXO1 on different DNA flap structures, we determined factors essential for the thermodynamic stability of EXO1. We show that enzymatic activity and stability of EXO1 on DNA is modulated by temperature. By characterization of EXO1 flap activity using various DNA flap substrates, we show that EXO1 has a strong capacity for degrading double stranded DNA and has a modest endonuclease or 5' flap activity. Furthermore, we report novel mechanistic insights into the processing of flap structures, showing that EXO1 preferentially cleaves one nucleotide inwards in a double stranded region of a forked and nicked DNA flap substrates, suggesting a possible role of EXO1 in strand displacement.

Keywords: EXO1; double stranded breaks; flap activity; strand displacement; threading; tracking.

Figure 1. Overview of EXO1 variants and purified full-length EXO1

(A) Schematic overview of full-length EXO1 and HEX-N2. Binding domains to MLH1, and MSH2 indicated at the COOH-terminus of full-length EXO1. In both full-length EXO1 and HEX-N2, the nuclease domain contain two domains, N and I domain, separated by a spacer. (B) SDS/PAGE analysis of purified EXO1. SDS/PAGE (8% gel). Lane 1=molecular weight marker (size in kDa on the left). Lane 2=purified EXO1.

Figure 2. Temperature modulates EXO1 activity

(A) Heat inactivation of EXO1 at 37°C. Representative gel of three independent biological replicas showing nuclease activities on a 5′ radioactive labelled 42-mer dsDNA GK142/GK143 (star denotes 5′-radiolabelling site), by incubation with recombinant purified EXO1. Lane 1 is substrate control, no EXO1 enzyme was added. Lanes 2–4 and lanes 5–7, both contain an increased amount of EXO1 0.5–1.5 pmol. Lanes 2–4 were pre-incubated at 4°C for 30 min prior the setup of the EXO1 assay (37°C for 30 min), acting as control for full activity. Lanes 5–7 were pre-heated for 30 min at 37°C, prior the EXO1 assay (37°C for 30 min). (B) Student t-test of EXO1 nuclease activity at 4°C and 37°C. Graph demonstrates that pre-incubation of EXO1 at 37°C results in a significant decrease in exonuclease activity, relative to incubation at 4°C (*P<0.05, **P<0.01), by comparing exonuclease product per EXO1 concentration. (C) Lower temperature increases EXO1 activity. Representative gel of four independent biological experiments showing that nuclease activities on a 5′-radioactive labelled 42-mer dsDNA (GK142/GK143) (star denotes 5′-radiolabelling site), by incubation with recombinant purified EXO1. Lane 1 is DNA substrate control, no EXO1 enzyme was added. Lanes 2–10, show EXO1 assay incubated with three different temperatures, respectively 25°C, 30°C, and 37°C, with increased concentration of EXO1 (0.5–1.5 pmol). (D) Student t-test of EXO1 nuclease activity at 25°C and 30°C versus 37°C. The EXO1 activity at 25°C was significantly increased in exonuclease activity compared with 37°C (*P<0.05). At 30°C, EXO1 only showed a significant increase in activity at 1 pmol versus at 37°C (*P<0.05). (E) Time course of EXO1 activity. Representative gel of three independent biological experiments showing that nuclease activities on a 5′-radioactive labelled 42-mer dsDNA GK142/GK143 (star denotes 5′-radiolabelling site), by incubation at time point, respectively 18.8, 37.5, 75, 150 s, 5, 10, 15, 30, 45, and 60 min with of EXO1 (2 pmol). (F) Graph shows analysis of EXO1 nuclease activity at different time points between 18.8 s and 60 min. The Student t-test indicated that there is no significant difference (P<0.05) between exonuclease product from the time points 10, 15, 30, 45, and 60 min.

Figure 3. EXO142-mer dsDNA substrate yield in a 12 bp product

The gel image showing EXO1 exonuclease activity on a 3′ labelled DNA substrate. Representative gel of three independent biological experiments showing that nuclease activities on a 3′-radioactive labelled 42-mer dsDNA (GK142/GK143) (star denotes 3′-radiolabelling site), by incubation with recombinant purified EXO1. In lane 1, a radioactive labelled marker was used to indicate 12 bp (GK192). In lanes 2–4, dsDNA substrate, 3′ labelled was used. In lane 2, no EXO1 enzyme was added. In lanes 3 and 4, 2 and 4 pmol EXO1 was added, respectively, and the oligo was degraded for 30 min at 30°C.

Figure 4. Assaying EXO1 endonuclease activity on different forked substrates

(A) Schematic overview of EXO1 processing a forked DNA substrate. The classically used forked substrate can be processed by endonuclease activity at the 5′-flap and exonuclease activity at the dsDNA end. EXO1 is shown in brown, red arrow points to the forked substrate sites of action (star denotes 5′-radiolabelling site). (B) The gel image shows EXO1 activity on four flap substrates differing in the length of dsDNA duplex. Representative gel of three independent biological experiments, showing endonuclease activity on a forked substrate 5′-radioactive labelled at flap (star denotes 5′-radiolabelling site). Lane 1 control, no EXO1 enzyme was added. Lanes 2–4 contains the classical flap substrate GK145/GK135 (21 bp duplex), lanes 5–7 contains flap substrate GK230/GK135 (16 bp duplex), lanes 8–11 GK229/GK135 (12 bp duplex), lanes 12–14 (8 bp DNA duplex) with increased concentration of EXO1 (0.5–1.5 pmol). Shortening of the duplex leads to reduced flap activity. (C) The gel image shows EXO1 flap activity on three fork substrates. Representative gel of three independent biological experiments showing nuclease activities on three different 5′-radioactive labelled flap substrates (star denotes 5′-radiolabelling site). Lane 1, no EXO1 enzyme was added, substrate control. Lanes 1–3 contain the classical forked DNA substrate GK145/GK135 with increased concentration of EXO1 (0.5–1.5 pmol). In lanes 4–6 the dsDNA end was 5′ blocked with a biotin. In lanes 7–9 the dsDNA end was 5′ blocked with a biotin-streptavidin. Blocking of the 5′ dsDNA end of the fork substrate led to decreased flap activity. (D) EXO1 flap activity on two forked and one hairpin DNA substrate. Representative gel of three independent biological experiments showing nuclease activities on a 5′-radioactive labelled at the flap. Lane 1, DNA substrate control, no EXO1 enzyme was added (GK145/GK135). Lanes 2–4 contain forked DNA substrate GK145/GK135, with increasing amount of EXO1 enzymes (0.5–1.5 pmol). Lanes 5–7 contain the GK145/GK144 DNA substrate 5′ biotin blocked at the dsDNA end. Lanes 8–10 contain a hairpin DNA substrate GK232, with increasing amounts of EXO1 enzyme.

Figure 5. EXO1 activity on a forked DNA substrate

(A) The gel image shows EXO1 activity on flap substrates. Lanes 1 and 2 contain a 5′ label DNA substrate GK247 in absence and presence of EXO1 (star denotes 5′-radiolabelling site). Lane 3 contains 5′ label 10 nt oligo (GK245). (B) Schematic overview of EXO1 processing hairpin forked DNA substrate. The forked substrate with a 10 nt flap is cleaved 1 bp inwards at the dsDNA resulting in 11 nt and smaller product of 9 nt (indicated with grey arrow). The 1 nt cleavage position at the ssDNA is indicated with a red arrow. (C) EXO1 process short flaps efficiently. Representative gel of five independent biological repeats of EXO1 showing nuclease activities on 5′-flap DNA substrates in length between 1 and 20 nt 5′ flap (star denotes 5′-radiolabelling site). DNA oligos GK251–GK253 are in range of 1 nt–5 nt 5′ flap and GK246 and GK249 contain respectively 9 nt and 20 nt 5′-flap. The different DNA substrates are setup in absence and presence of EXO1. (D) Analysis of the distribution of substrate and degraded flap and 1 nt product. (E) Statistical analysis of the degradation of the flap substrates. One-way analysis of variance with Tukey's multiple-comparison test of the EXO1 nuclease activity in salt condition. Percent of EXO1 activity on different 5′-flap DNA substrates. The 1 and 2 nt DNA 5′-flap are both significantly (***P<0.001) more degraded than longer flaps (>2 nt).

Figure 6. Tracking or threading of EXO1 along the ssDNA flaps to junction

(A) Representative gel of five independent biological experiments showing that EXO1 detects the 5′ DNA 20-mer flap by tracking and threading. Lanes 1–2 contain substrate GK249, lanes 3–4 substrate GK250, which contain a hairpin loop in the ssDNA (starting at nucleotide position 6 from the 5′ end), and lanes 5–6 substrate GK254 which contains a TEG-biotin, at position 6 nt from 5′ end on the ssDNA and lanes 7–8 substrate GK254 contains in presents of streptavidin. Lanes 2, 4, 6 and 8 are substrates in the presence of EXO1. (B) Statistical analysis of degradation of the different DNA conversions. One-way analysis of variance with Tukey's multiple-comparison test. GK250 shows a trend in versus GK249. GK254 and GK249 show a similar degradation. (C) A schematic model of tracking, where EXO1 detect the junction of the 5′ flap and cleave directly. (D) A schematic model of threading, where EXO1 detects the ssDNA end at the 5′ flap and slide of the ssDNA to the junction of the 5′ flap and cleave.

Figure 7. Fork, gap and nick DNA configurations are differently processed by EXO1

(A), (B) and (C) Representative gels of at least three independent biological experiments showing EXO1 catalysing forked, gap and nick DNA substrates with a 5′ flap of (A) 1 nt, (B) 5 nt and (C) 10 nt. (D) Statistical analysis of degradation of fork, gap and nick substrate of different length by EXO1. One-way analysis of variance with Tukey's multiple-comparison test with significance. The letters indicate F=fork, G=gap and N=nick substrate, ns=not significant. Stars in the table indicated the significance (P<0.05) between the different parameters.