Liao S , Toczylowski T , Yan H .

R01 GM57962-02 NIGMS NIH HHS

	Figure 1. Establishment of cytosol as a system for studying 5′- strand processing. (A) A dideoxynucleotide at the 3′-end blocks NHEJ. A 5.7 kb linear DNA (pBLP) with either dC or ddC at the 3′- end was incubated in cytosol at 5 ng/µl. Samples were taken at the indicated times, treated with SDS–proteinase K and separated on a 1% TAE–agarose gel. DNA was detected by SYBR Gold staining. L: linear substrate. S: supercoiled monomer product. Bracket: dimers, trimers and multimers. (B) Effect of DNA concentration on 5′-strand processing. DNA with ddC-terminated 3′-ends was incubated in cytosol at 5 ng/µl and 1 ng/µl concentrations. Samples were processed as in (A), transferred to a Nylon membrane, and detected by Southern hybridization with a 32P-labeled probe. L: liner substrate. Bracket: intermediates. (C) Differential fate of 5′- and 3′ 32P-label during 5′ strand processing. 5′- or 3′-labeled DNA with ddC-terminated 3′-ends was incubated in cytosol for the indicated times and separated by gel electrophoresis as in (A). The gel was dried and the 32P signal was detected by exposure to an X-ray film.
	Figure 2. Characterization of xEXO1's nuclease activity. (A) A SDS–PAGE showing the purified recombinant wild-type and mutant xEXO1. (B) Nuclease assay of recombinant xEXO1 with different DNA substrates. The substrates were 48-mer oligonucleotides in either ss- or ds-form attached to magnetic beads, leaving the 5′-end (of the 32P-labeled strand) accessible to the nuclease. After 1 h of incubation at room temperature, the reactions were terminated with 1% SDS, heated at 95°C for 15 min, and then separated on an 8% TAE–PAGE. (C) Effect of RPA on the 5′→3′ exonuclease activity of xEXO1. Different amounts of RPA and xEXO1 were incubated with the 32P-labeled 5′ accessible ss- or ds-48-mer beads for one hour at room temperature. The analysis was similar to that in (A). (D) Determination of the 3′→5′ exonuclease activity of xEXO1. Four microliters of xEXO1 was incubated with 32P-labeled 3′ accessible ss- or ds-48-mer beads in the presence or absence of RPA (75 nM) at room temperature for 1 h. The analysis was similar to that in (A).
	Figure 3. Effect of xEXO1 on 5′-strand processing. (A) Detection of xEXO1 in cytosol and NPE by western blot with anti-xEXO1 bleed (immune) and pre-bleed (pre-immune). (B) Efficiency of xEXO1 depletion. The quantitation standards for western blot were untreated cytosol at different amounts relative the depleted cytosol. (C) Effect of xEXO1 depletion on 5′-strand processing. 3′-32P-labeled linear pBLP DNA with ddC-blocked ends (1 ng/µl) was incubated in xEXO1-depleted or mock-depleted cytosol. Four additional reactions of xEXO1-depleted cytosol were supplemented two different levels of wild-type (wt) and mutant (mt) recombinant xEXO1. Samples taken at the indicated times were treated with SDS–proteinase K and separated on 1% TAE–agarose gel. The gel was stained with SYBR Gold and then dried for exposure to an X-ray film.
	Figure 4. Effect of xEXO1 and xDNA2 co-depletion on 5′-strand resection. (A) Efficiency of xEXO1 and xDNA2 depletion. The quantitation standards for western blot were untreated cytosol at different amounts relative the depleted cytosol. (B) Effect of xDNA2 single depletion on 5′-strand processing in cytosol. The 3′-32P-labeled linear pBLP DNA with ddC-blocked ends (1 ng/µl) was incubated in mock-depleted cytosol or xDNA2-depleted cytosol (supplemented with either the ELB buffer or the purified xDNA2). Samples were taken at the indicated times, treated with SDS–proteinase K and analyzed on a 1% TAE–agarose gel. (C) Effect of xEXO1 and xDNA2 double depletion on 5′-strand processing. Cytosol depleted of the indicated proteins was incubated with the 3′-labeled ddC-blocked linear pBLP (1 ng/µl). Samples taken at the indicated times were treated with SDS–Proteinase K, separated on an 1% TAE–agarose gel, transferred to a Nylon membrane, and detected by Southern hybridization with a 32P-labeled probe. (D) Plot of the substrate remaining at the indicated times after incubation in the various depleted extract. The averages and standard deviations were derived from three sets of data.
	Figure 5. Effect of xEXO1 and xDNA2 on 3′-labeled ssDNA degradation. Linear ss-pBS DNA (2 ng/µl) labeled with 32P at the 3′-end was incubated in the indicated depleted cytosol or buffer. Samples taken at the indicated times were treated with SDS–proteinase K, separated on a 1% TAE–agarose gel, and detected by exposure to an X-ray film.
	Figure 6. Effect of xEXO1 and xWRN co-depletion on 5′-strand processing. (A) Western showing the depletion of xEXO1, xWRN, and xDNA2. (B) Effect of xWRN single depletion on 5′-strand processing. The 3′-32P-labeled linear pBLP DNA with ddC-blocked ends (1 ng/µl) was incubated in mock-depleted cytosol or xWRN-depleted cytosol (supplemented with either buffer or the purified xWRN). Time points were treated with SDS–proteinase K and analyzed on a 1% TAE–agarose gel. (C) Effect of xEXO1-xWRN and xDNA2-xWRN double depletions on 5′-strand processing. Cytosol depleted of the indicated proteins were incubated with the 3′-32P-labeled and ddC-blocked linear pBLP (1 ng/µl). The reactions were treated and analyzed similarly to that in (B). The percentage of remaining DNA substrate was calculated relative to the total input DNA.
	Figure 7. Effect of xEXO1 and xWRN on the initiation of 5′-strand processing. The 5′-32P-labeled linear pBLP DNA with ddC-blocked ends (1 ng/µl) was incubated with cytosol depleted of the indicated proteins. Samples were taken at the indicated times, treated with SDS–proteinase K, and analyzed by a 1% TAE–agarose gel. The percentage of 5′-32P-label retained on the substrate was calculated relative the total input.
	Figure 8. Model for the 5′-strand-specific processing of DNA ends. xWRN-xDNA and xEXO1 constitute two parallel pathways. MRN and CtIP act with xWRN-xDNA2 and xEXO1 in the initiation step, but are dispensable after partial degradation of the 5′-strand.

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