The Bloom's and Werner's syndrome proteins are DNA structure-specific helicases - PubMed (original) (raw)

The Bloom's and Werner's syndrome proteins are DNA structure-specific helicases

P Mohaghegh et al. Nucleic Acids Res. 2001.

Abstract

BLM and WRN, the products of the Bloom's and Werner's syndrome genes, are members of the RecQ family of DNA helicases. Although both have been shown previously to unwind simple, partial duplex DNA substrates with 3'-->5' polarity, little is known about the structural features of DNA that determine the substrate specificities of these enzymes. We have compared the substrate specificities of the BLM and WRN proteins using a variety of partial duplex DNA molecules, which are based upon a common core nucleotide sequence. We show that neither BLM nor WRN is capable of unwinding duplex DNA from a blunt-ended terminus or from an internal nick. However, both enzymes efficiently unwind the same blunt-ended duplex containing a centrally located 12 nt single-stranded 'bubble', as well as a synthetic X-structure (a model for the Holliday junction recombination intermediate) in which each 'arm' of the 4-way junction is blunt-ended. Surprisingly, a 3'-tailed duplex, a standard substrate for 3'-->5' helicases, is unwound much less efficiently by BLM and WRN than are the bubble and X-structure substrates. These data show conclusively that a single-stranded 3'-tail is not a structural requirement for unwinding of standard B-form DNA by these helicases. BLM and WRN also both unwind a variety of different forms of G-quadruplex DNA, a structure that can form at guanine-rich sequences present at several genomic loci. Our data indicate that BLM and WRN are atypical helicases that are highly DNA structure specific and have similar substrate specificities. We interpret these data in the light of the genomic instability and hyper-recombination characteristics of cells from individuals with Bloom's or Werner's syndrome.

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Figures

Figure 1

Figure 1

Unwinding of 32P-labelled substrates by WRN. Reactions contained 20 mM Tris–HCl, pH 7.5, 2 mM MgCl2 2 mM ATP, 0.1 mg/ml BSA, 1 mM DTT, 32P-labelled substrate and WRN. Substrates were: (A) 0.5 nM nicked duplex incubated with 10 nM WRN; (B) 1 nM 50 bp blunt-ended duplex incubated with 7.3 nM WRN; (C) 1 nM 4 bp bubble incubated with 1 nM WRN; (D) 1 nM synthetic X-junction incubated with 1.4 nM WRN. Lane +, boiled substrate; lane –, time 0; lane 1, 1 min; lane 2, 2 min; lane 3, 5 min; lane 4, 10 min; lane 5, 20 min; lane 6, 30 min incubation at 37°C. The positions of the starting substrate (dsDNA) and single-stranded DNA products (ssDNA) are indicated on the right. For the X-junction, the positions of the 4-way junction substrate and 2-way junction (splayed arm) and ssDNA products are indicated.

Figure 2

Figure 2

Time course of unwinding of selected DNA substrates by BLM and WRN. Reactions were performed essentially as described in the legend to Figure 1, using 1 nM substrate in each case and 20 nM BLM (set of reactions on the left, as indicated above) or 10 nM WRN (reactions on the right). WRN reactions on the bubble substrate contained 5 nM enzyme to minimise loss of substrate, which was particularly susceptible to exonucleolytic degradation. (A–D) Results for the synthetic 4-way junction, forked duplex, 3′-tailed duplex and 12 bp bubble substrate, respectively. Lanes 1–6 depict a time course of 0, 2, 4, 8, 12 and 18 min incubation at 37°C. The positions of the substrates and reaction products are indicated on the right.

Figure 3

Figure 3

WRN can unwind a variety of G4 DNA substrates. (A) Time course of unwinding of 1 nM G4-TP substrate by 5 nM WRN. (B) Time course of unwinding of 1 nM G4-OX-IT substrate by 1 nM WRN. (C) Time course of unwinding of 1 nM G4-OX-1 substrate by 1 nM WRN. Lane +, boiled substrate; lane –, no enzyme; lanes 1–6, times of incubation at 37°C of 1, 2, 5, 8, 16 and 24 min, respectively. The positions of the G-DNA substrate and the ssDNA products are indicated on the right. (D) Quantification of the data from (B) and (C), comparing the rates of unwinding of the G4-OX-1T substrate containing a 7 nt 3′-tail and the equivalent G4-OX-1 substrate lacking the tail.

Figure 3

Figure 3

WRN can unwind a variety of G4 DNA substrates. (A) Time course of unwinding of 1 nM G4-TP substrate by 5 nM WRN. (B) Time course of unwinding of 1 nM G4-OX-IT substrate by 1 nM WRN. (C) Time course of unwinding of 1 nM G4-OX-1 substrate by 1 nM WRN. Lane +, boiled substrate; lane –, no enzyme; lanes 1–6, times of incubation at 37°C of 1, 2, 5, 8, 16 and 24 min, respectively. The positions of the G-DNA substrate and the ssDNA products are indicated on the right. (D) Quantification of the data from (B) and (C), comparing the rates of unwinding of the G4-OX-1T substrate containing a 7 nt 3′-tail and the equivalent G4-OX-1 substrate lacking the tail.

Figure 4

Figure 4

Comparative unwinding activity of the BLM and WRN helicases on different DNA substrates. (A) Rates of the unwinding reaction (nM substrate/µM protein/min) were derived as described in Materials and Methods. (B) k is the pseudo first order rate constant of DNA unwinding, which was calculated as described in Materials and Methods. Solid bars, BLM; hatched bars, WRN. The substrates are indicated along the horizontal axis: G4, OX-1T G-quadruplex; HJ, X-junction; fork, forked duplex; bub-12, duplex with 12 bp bubble; 3′-tail, 3′-tailed duplex; nick, nicked duplex; blunt, 50 bp duplex; bub-4, duplex with 4 bp bubble; 5′-tail, 5′-tailed duplex.

Figure 4

Figure 4

Comparative unwinding activity of the BLM and WRN helicases on different DNA substrates. (A) Rates of the unwinding reaction (nM substrate/µM protein/min) were derived as described in Materials and Methods. (B) k is the pseudo first order rate constant of DNA unwinding, which was calculated as described in Materials and Methods. Solid bars, BLM; hatched bars, WRN. The substrates are indicated along the horizontal axis: G4, OX-1T G-quadruplex; HJ, X-junction; fork, forked duplex; bub-12, duplex with 12 bp bubble; 3′-tail, 3′-tailed duplex; nick, nicked duplex; blunt, 50 bp duplex; bub-4, duplex with 4 bp bubble; 5′-tail, 5′-tailed duplex.

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