Liao S , Guay C , Toczylowski T , Yan H .

GM57962-02 NIGMS NIH HHS , P30 CA006927 NCI NIH HHS

	Figure 1. Depletion of MRE11 blocks 5′-strand resection. (A) Detection of MRE11 in cytosol and NPE by Western blot with anti-Xenopus MRE11 antibodies. (B) Efficiency of MRE11 depletion from cytosol. The standards for quantitation were cytosol loaded at different amounts relative to the depleted cytosol. (C) Effect of MRE11 depletion on 5′-strand resection. 3′-32P-labeled (at the underlined A) linear pBLP DNA with ddC-blocked ends (1 ng/ul) was incubated in the MRE11-depleted or mock-depleted cytosol. Samples were taken at the indicated times, treated with SDS/proteinase K and separated on a 1% TAE/agarose gel. The gel was first stained with SYBR Gold and then dried for exposure to an X-ray film. (D) Effect of MRE11 depletion on the initiation of 5′-strand processing. The 5′-32P-labeled linear pBLP DNA with ddC-blocked ends (1 ng/ul) was incubated with the MRE11-depleted or mock-depleted cytosol. Samples taken at the indicated times were analyzed as in (C). (E) Plot of the amounts of 3′- and 5′-32P on the remaining substrates at the indicated times after incubation in the MRE11-depleted or mock-depleted extracts. The averages and standard deviations were calculated with three sets of data.
	Figure 2. Complementation of MRE11 depletion by the purified MRN protein. (A) A SDS-PAGE showing the final gel-filtration fractions of the purified Xenopus MRN complex. The gel was stained with Coomassie blue and the staining intensity of fraction #9 was plotted. (B) Complementation of MRE11 depletion with the purified MRN complex. The 3′-labeled linear pBLP DNA substrate was incubated in the MRE11-depleted cytosol supplemented with MRN or buffer. Samples taken at the indicated times were analyzed as in Figure 1C. (C) Plot of the complementation assays. The 3′-32P on the remaining substrate DNA at the indicated times from three experiments were used to calculate the averages and standard deviations.
	Figure 3. CtIP is important for 5′-strand resection but can be bypassed by excess MRN. (A) Efficiency of CtIP depletion. The standards for quantitation were cytosol loaded at different amounts relative to the depleted cytosol. (B) Effect of CtIP depletion on 5′-strand resection. 3′-32P-labeled linear pBLP DNA with ddC-blocked ends (1 ng/ul) was incubated in the CtIP-depleted or mock-depleted cytosol. Samples taken at the indicated times were analyzed by agarose gel electrophoresis. (C) Effect of CtIP depletion on the removal of the 5′-32P label. Samples taken at the indicated times were analyzed by agarose gel electrophoresis. (D) Complementation of CtIP depletion with excess MRN. 3′-labeled substrate was incubated in the CtIP-depleted cytosol supplemented with MRN (2x the endogenous level = ca. 16 ng/ul) or buffer. Samples taken at the indicated times were analyzed by agarose gel electrophoresis. (E) Plot of the complementation assays. The amounts of 3′-32P on the remaining substrates at the indicated times from three experiments were used to calculate the averages and standard deviations.
	Figure 4. Excess MRN cannot bypass WRN and EXO1. (A) Effect of excess MRN on 5′-strand processing in WRN and EXO1 co-depleted cytosol. The 3′-32P-labeled pBLP DNA with ddC-blocked ends (1 ng/ul) was incubated in the mock-depleted cytosol or WRN and EXO1 co-depleted cytosol [supplemented with buffer, EXO1 or MRN (2x)]. Samples taken at the indicated times were treated with SDS/proteinase K and analyzed on a 1% TAE-agarose gel. (B) Effect of excess MRN on the removal of the 5′-32P label in WRN and EXO1 co-depleted cytosol. 5′-labeled pBLP (1 ng/ul) was incubated in mock-depleted cytosol or WRN and EXO1 co-depleted cytosol (supplemented with buffer or 2x MRN). Samples were treated and analyzed similarly to that in A.
	Figure 5. MRE11 and CtIP can be bypassed by a 3′-ss-tail. (A) 3′-labeled DNA (at the underlined nucleotide), either blunt ended or with a 3′-ss-tail was incubated with the indicated depleted cytosol. Samples taken at the indicated times were analyzed by TAE-agarose and the products were detected by exposure to an X-ray film. (B) Plot of the kinetics of 5′-32P removal by the purified MRN (2x) and the kinetics of 3′-labeled DNA degradation in the CtIP-depleted cytosol supplemented with MRN (2x). Three sets of data were used to calculate the averages and standard deviations.
	Figure 6. Purified MRN, WRN, DNA2 and RPA are sufficient to reconstitute 5′-strand resection. (A) MRN stimulated the activity of WRN-DNA2-RPA towards blunt-ended DNA. WRN, DNA2 and RPA were incubated with 3′-labeled DNA in the presence or absence of MRN. Samples taken at the indicated times were separated on a 1% TAE-agarose and the products were detected by exposure to an X-ray film. (B) The effect of each component on the reconstituted MRN-WRN-DNA2-RPA pathway. Various combinations of proteins and ATP were incubated with the 3′-labeled blunt-ended DNA. Samples taken at the indicated times were analyzed similarly to those in A. A long exposure was also presented to show the faint products. (C) Interactions of MRN with DNA2 and RPA. MRN was incubated with DNA2 or RPA and then immunoprecipitated by anti-MRN antibodies. The beads and supernatants were analyzed by Western blot with antibodies against DNA2, RPA, and RAD50.
	Figure 7. MRN stimulates the activity of EXO1. (A) EXO1 and MRN, alone or together, were incubated with 3′-32P labeled blunt-ended DNA. Samples taken at the indicated times were separated on a 1% TAE-agarose and the products were detected by exposure to an X-ray film. (B) EXO1 and MRN, alone or together, were incubated with 3′-labeled DNA with a 3′-overhang (ca. 200-300 nt). Samples taken at the indicated times were separated on a 1% TAE-agarose and the products were detected by exposure to an X-ray film. (C) MRN helps recruit EXO1 to DNA ends. EXO1 and MRN, alone or together, were incubated with 1.5 kb DNA anchored via biotin at one end onto Streptavidin magnetic beads. A reaction with EXO1, MRN and blank beads was also set up as a control. The beads and supernatant fractions were analyzed for EXO1 and RAD50 by Western blot analysis.
	Figure 8. Model for the two pathways of 5′-strand resection. MRN is directly involved in the initiation of resection while CtIP stimulates the activity of MRN. The other resection proteins are involved in both the initiation step and the extension step.

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