Liao S, Tammaro M, Yan H.

P30 CA006927 NCI NIH HHS , R01 GM057962 NIGMS NIH HHS

	Figure 1. DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. (A) DNA substrates bearing different types of 3′ ends and labeled by 32P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. (B) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. (C) Assay for detecting biotin at the 3′ end of ss-DNA. The 32P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. (D) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.
	Figure 2. DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. (A) 32P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. (B) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. (C) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.
	Figure 3. Mre11 but not its nuclease activity is essential for the resection of DNA with 3′ damaged nucleotides or bulky adducts. (A) Effect of MRE11 depletion on the resection of 3′ ddC and 3′ avidin DNA. The substrates were incubated in mock-depleted or Mre11-depleted extracts (with or without 8 ng/μl wild-type (wt) and mutant (mt) MRN) and the products were analyzed on a 1% TAE-agarose gel. (B) Plots of the amounts of 3′ 32P on the remaining substrates at the indicated times. The averages and standard deviations were calculated with three sets of data. (C) A Coomassie blue stained SDS-PAGE gel showing the purified MRN complexes. (D) Ss-DNA endonuclease assay of the MRN complexes. Wild-type and mutant MRN (8ng/μl) were incubated with ss-M13 DNA (10 ng/μl) at 22°C for 30 min. The products were separated on a TAE-agarose gel and detected by SYBR Gold staining.
	Figure 4. The Mre11 nuclease activity is critical for the resection of DNA with 5′ bulky adducts. (A) Effect of Mre11 depletion on the resection of 5′ p-Tyr and 5′ avidin DNA. The substrates were incubated in mock-depleted or Mre11-depleted extracts and the products were analyzed on a 1% TAE-agarose gel. (B) Plots of the amounts of 32P on the remaining substrates at the indicated times. (C) Detection of 5′ biotin on the resection intermediates of 5′ avidin DNA. Control: untreated substrate. Mock and –Mre11: intermediates isolated after 30 min in the indicated extracts. They were pre-incubated with buffer or avidin, and then treated with T7 Exo. (T7 Exo falls off DNA once the two enzyme molecules meet in the middle, resulting in the accumulation of ss-DNA of the 3′ half). The reactions also contained a plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, and dried for exposure to X-ray film.
	Figure 5. The structure of ends affects the dependence on CtIP for resection. (A) Effect of CtIP depletion on 3′ avidin DNA resection. DNA was incubated in mock-depleted or CtIP-depleted extracts supplemented with 2x excess wild-type or mutant MRN (16ng/μl final concentration). (B) Effect of CtIP depletion on 5′ avidin DNA resection. DNA was incubated in mock-depleted or CtIP-depleted extracts supplemented with 2× excess wild-type MRN (16ng/μl final concentration). (C) Plots of the amounts of 3′ 32P on the remaining substrates at the indicated times. (D) Detection of 5′ biotin on the resection intermediates of 5′ avidin DNA. The intermediates were isolated after 30 minutes incubation and analyzed as in Figure 4.
	Figure 6. MRN can stimulate resection by an Mre11 nuclease-independent mechanism. (A) Effect of MRN on the activities of lambda exonuclease and Exo1 against 3′ avidin DNA. (B) Comparison of the wild-type and mutant MRN complexes on the stimulation of Exo1 activity against 3′ avidin DNA. (C) Plot of the amounts of 3′ 32P on the remaining substrates at the indicated times. The averages and standard deviations were calculated with three sets of data.
	Figure 7. Model for the repair pathway choice and Mre11 nuclease dependence of different types of ends. Normal ends are predominantly channeled to resection (except in S. cerevisiae). Ends with minor damages are preferentially channeled to resection. Ends with bulky adducts are exclusively channeled to resection. Resection is absolutely dependent on Mre11, but its nuclease activity and CtIP are essential only for ends with 5′ bulky adducts.

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