Liao S , Toczylowski T , Yan H .

R01 GM57962-02 NIGMS NIH HHS

	Figure 1. Purification and identification of the major 5′→3′ ssDNA exonuclease in Xenopus extracts. (A) Purification scheme. (B) Nuclease assay of the heparin flow through and 250 mM NaCl elute. The oligo substrate carried two 32P-labeled dA near the 3′-end and was attached to magnetic beads through a biotin at the 3′-end. After incubation at room temperature for 1 h, the reactions were terminated with 1% SDS, heated at 95°C for 5 min and then analyzed on an 8% TAE/PAGE. The 32P signal was detected by Phophoimager (Fuji). The nuclease activity was determined by calculating the fraction of small product generated (normalized to that of reaction 3 that contained both the 250 mM elute and the flow through). (C) A silver stained SDS–PAGE gel (4–12%) of the purified xDNA2 after the oligo beads purification. Load, proteins before binding to the oligo beads; unbound, proteins after incubation with beads; elute, bound proteins eluted by high salt buffer. (D) Nuclease assay of the purified xDNA2 in the presence of heparin flow through or buffer. The assay condition and the determination of the nuclease activity were similar to those in (B).
	Figure 2. Identification of xRPA as the stimulating factor for xDNA2. (A) Western blot of xRPA (p70 subunit) in the heparin flow through before and after incubation with ss-oligo beads. (B) The stimulating activity in heparin flow through, before and after incubation with oligo beads, and pure xRPA (at 90 nM and 30 nM). The reactions were terminated with 1% SDS, heated at 95°C for 5 min, and then analyzed on an 8% TAE/PAGE. (C) Nuclease assay with different DNA substrates. The substrates were 48-mers in either ss- or ds-form. They were attached to magnetic beads, leaving either the 5′ or the 3′-end (of the 32P-labeled strand) accessible to the nuclease. The assay condition was similar to that in (B).
	Figure 3. Effect of xDNA2 on DNA end processing. (A) Depletion efficiency, end processing substrate, and reaction set up. The quantitation standards for western analysis were NPE loaded at the indicated amounts relative the depleted NPE. The 3′-ends of the partially filled-in linear pUC19 were labeled with 32P-dA by Klenow. (B) Effect of xDNA2 depletion on end processing. DNA (2 ng/µl) was incubated in xDNA2-depleted NPE, mock-depleted NPE or xDNA2-depleted NPE supplemented with either ELB buffer or the purified xDNA2 protein (to ∼15% of the endogenous xDNA2 level) in the presence of ddNTPs (to block NHEJ). Two additional reactions containing xDNA2 or ELB buffer served as controls. Samples were taken at the indicated time, treated with SDS/proteinase K and separated on a 1% TAE/agarose gel. The gel was first stained with SYBR Gold to detect total DNA and then dried for exposure to X-ray film to detect 32P.
	Figure 4. Effect of xDNA2 on 5′ ss-tail degradation. Denatured pUC19 DNA (2 ng/µl) labeled at the 3′-end was incubated in xDNA2-depleted or mock-depleted NPE supplemented with buffer or the purified xDNA2 (to ∼5% of the level of the endogenous xDNA2). Time points were treated with SDS/proteinase K and separated on a 1% TAE agarose gel. The gel was first stained with SYBR Gold to detect total DNA and then dried for exposure to X-ray film to detect 32P. The band migrated just below the ss-pUC19 DNA is the RNA in NPE.
	Figure 5. Effect of xDNA2 on end unwinding. (A) Principle of the unwinding assay. Thin line: normal nucleotides; thick line: thio nucleotides; *, 32P label. (B) The thio 5′ oligo duplex precoated onto Streptavidin magnetic beads was incubated in xDNA2-depleted or mock-depleted NPE. Samples were separated into bead and supernatant fractions, treated with SDS/proteinase K and analyzed on a 10% native TAE PAGE. B, beads; S, supernatant. The SDS/proteinase K treatment in this study was at room temperature for 2 h, a condition not harsh enough to disrupt the binding of biotin with Streptavidin (1).
	Figure 6. Effect of xDNA2 on SSA. The substrate, pRW4′, which carried two direct repeats at the partially filled-in XhoI protruding ends, was incubated in mock-depleted or xDNA2-depleted NPE (supplemented with xDNA2 or buffer) at room temperature. DNA samples were taken at the indicated times, treated with SDS/EDTA/Proteinase K, separated by TAE/agarose gel electrophoresis and detected by SYBR Gold stain. The SSA products include the band indicated by the arrow and a subset of the bands indicated by the bracket. The asterisks indicate the NHEJ products.
	Figure 7. Model for the strand-specific processing of DNA ends. (A) End processing in the nucleoplasmic extract. xWRN (and potentially other helicases) are recruited to unwind the end in an ATP-dependent reaction. xDNA2 (and another exonuclease(s)) degrades the 5′ ss-tail. Proteins like MRE11 and CtIP (not depicted) might act upstream of xWRN. (B) The postulated RecQ-RecJ mechanism for end processing.

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