Modulating the body's networks could become mainstream therapy for many health issues.
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Communicated by Stanley M. Gartler, University of Washington, Seattle, WA (received for review December 15, 1999)
Article Figures & SI
Figures
- Figure 1
WRN does not increase primer extension by DNA polymerase α or ɛ. A 5′-end labeled 14-nt DNA primer was hybridized to a 46-nt DNA template. The primer (0.1 pmol) was extended by DNA polymerase α or ɛ in the absence or presence of increasing concentrations of WRN (1.2–60 fmol). The reactions were incubated at 37°C for 10 min and were terminated by the addition of an equal volume of denaturing loading buffer. Aliquots were electrophoresed through 14% polyacrylamide-urea gels, and extension products were visualized by autoradiography. The panel labeled “(−) Pol” is a control of increasing concentrations of WRN incubated with the primer/template in the absence of DNA polymerase to demonstrate the products of the 3′→5′ exonucleolytic activity of WRN.
- Figure 2
WRN increases primer extension by DNA polymerase δ. Indicated amounts of pol δ were mixed with 0.1 pmol of the 14/46 primer/template in the absence or presence of a fixed concentration of WRN (≈6 fmol/10 μl reaction). Reactions were incubated at 37°C for 10 min and were processed as described in the legend to Fig. 1. Lanes: 1, substrate alone; 2, primer/template incubated with ≈6 fmol WRN in the absence of pol δ.
- Figure 3
WRN stimulates the rate of incorporation of the initiating nucleotide by DNA pol δ. 0.5 pmol of the 14/46 primer/template was incubated with ≈0.3 fmol of pol δ in the absence (−) or presence (+) of 24 fmol of WRN. dGTP was included at concentrations ranging from 0.02–5 μM, and the reactions were incubated for either 5 min [(−)WRN] or 3 min [(+)WRN] at 37°C. The reactions were quenched by the addition of denaturing loading buffer and were electrophoresed through 14% polyacrylamide-urea gels as described in Materials and Methods. The extension products were visualized by autoradiography (A), and the amounts of 15-mer generated were quantitated by PhosphorImager analysis. The kinetic constants were derived from Hanes-Woolf plots of the data (B).
- Figure 4
WRN does not increase the activity of DNA polymerase δ holoenzyme. 0.125 pmol of singly primed single-stranded mp18 DNA was coated with E. coli single-stranded DNA binding protein and was loaded with PCNA (0.5 pmol) by replication factor C (0.125 pmol). DNA synthesis was initiated by the addition of pol δ (0.125 pmol) in the absence or presence of WRN (0.25 pmol). Reactions were incubated at 13°C; aliquots (12 μl) were removed at 10, 20, and 40 min after incubation and were electrophoresed through alkaline agarose gels. Extension products were visualized by autoradiography of the dried gel. Positions of migration of size markers, expressed in kilobases, are indicated on the left.
- Figure 5
Functional interaction with WRN requires the Pol32p subunit of S. cerevisiae DNA pol δ. Pol δ (≈0.3 fmol), pol δ* (≈0.9 fmol), or in vitro reconstituted pol δ [Pol δ*+Pol32p] (≈0.6 fmol)] were incubated with the 14/46 primer/template such that comparable amounts of primer were extended in each case. Each preparation of polymerase was incubated with increasing amounts of WRN (2.4–60 fmol); assays were carried out and processed as described in the legend to Fig. 1.
Functional interaction between the Werner Syndrome protein and DNA polymerase δ
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