Mobile D-loops are a preferred substrate for the Bloom's syndrome helicase

Abstract

The Bloom's syndrome helicase, BLM, is a member of the highly conserved RecQ family, and possesses both DNA unwinding and DNA strand annealing activities. BLM also promotes branch migration of Holliday junctions. One role for BLM is to act in conjunction with topoisomerase IIIalpha to process homologous recombination (HR) intermediates containing a double Holliday junction by a process termed dissolution. However, several lines of evidence suggest that BLM may also act early in one or more of the recombination pathways to eliminate illegitimate or aberrantly paired DNA joint molecules. We have investigated whether BLM can disrupt DNA displacement loops (D-loops), which represent the initial strand invasion step of HR. We show that mobile D-loops created by the RecA recombinase are a highly preferred substrate for BLM with the invading strand being displaced from the duplex. We have identified structural features of the D-loop that determine the efficiency with which BLM promotes D-loop dissociation. We discuss these results in the context of models for the role of BLM as an 'anti-recombinase'.

Figure 1

Schematic representation of the different D-loop molecule species used in this study. The radiolabelled invading strand is shown in green. The triangles denote the 3′ end, while the squares denote the 5′ end of each oligonucleotide. The position of the radioactive label is indicated by a red asterisk.

Figure 2

Kinetics of unwinding of oligonucleotide-based D-loops in a time-course experiment. Unwinding was carried out in the presence of 0.64 nM BLM for the period of time indicated above the lanes. The position of the substrate and fully unwound single-stranded oligonucleotide products, together with reaction intermediates A and B, are shown on each side. The flame symbol denotes heat-denatured DNA.

Figure 3

A plasmid-based, mobile D-loop is a substrate for BLM. (a) BLM-concentration dependent unwinding of pDL3 in a 15 min reaction. The enzyme concentration was varied (as indicated by the triangle above) between 0.7 and 5.6 nM in steps of 0.7. ‘0’ denotes a control sample in the absence of BLM, which was otherwise treated identically. (b) Unwinding of pDL3 in a time-course experiment. Samples were taken at the time points indicated above the lanes. The positions of the substrate and the free radiolabelled oligonucleotide are indicated on the left.

Figure 4

Comparison of the efficiency of unwinding of different substrates by BLM. BLM enzyme-concentration dependent unwinding of the pDL3 mobile D-loop (upper panel), the X12 four-way junction (middle panel) and G4 (lower panel) substrates. The enzyme-concentration was varied between 1–40 nM as indicated above by the triangle. Reactions lacking BLM are denoted by ‘−’ above. The flame symbol denotes heat-denatured DNA. The substrates and the products of unwinding are shown on the left of each panel. (b) Quantification of the data from (a). Blue triangle, green diamond and red square symbols depict the pDL3, four-way junction and G4 DNA substrates, respectively. Analysis of the concentration-dependence curve indicated that 50% unwinding of pDL3, four-way junction and G4 DNA is achieved at 7.38, 8.97 and 5.84 nM, respectively. At high concentrations of BLM, the strand annealing activity of the enzyme becomes dominant over the unwinding activity, resulting in a diminution in the level of unwound product for the four-way junction substrate [middle panel of (a), green triangles and dotted line of (b)]. The last two concentration points (30 and 40 nM) were, therefore, omitted from the non-linear regression analysis and for determination of the 50% unwinding concentration (see Materials and Methods).

Figure 5

Competition of BLM-dependent unwinding of different substrates. (a) Competition with a cold, unrelated, 80 nt oligonucleotide in reactions containing the pDL3 D-loop (upper panel), the X12 four-way junction (middle panel) and G4 DNA (lower panel). Lanes marked ‘0’ contained BLM but no competitor. (b) % inhibition expressed as a function of inhibitor concentration. A total of 50% inhibition of unwinding of pDL3, four-way junction and G4 was achieved at 29.8, 2.73 and 29.88 nM, respectively. (c) Competition with cold four-way junction. (d) % inhibition expressed as a function of inhibitor concentration. A total of 50% inhibition of unwinding of the radiolabelled four-way junction substrate occurred at 1.91 nM. No significant inhibition of unwinding of either the plasmid-based D-loop or G4 DNA was found in the concentration range used. (e) Competition with cold G4 DNA. (f) % inhibition expressed as a function of inhibitor concentration. A total of 50% inhibition of unwinding of the pDL3 (blue triangle) and G4 DNA (red square) substrates occurred at 20.78 and 30.39 nM concentration, respectively.

Figure 5

Competition of BLM-dependent unwinding of different substrates. (a) Competition with a cold, unrelated, 80 nt oligonucleotide in reactions containing the pDL3 D-loop (upper panel), the X12 four-way junction (middle panel) and G4 DNA (lower panel). Lanes marked ‘0’ contained BLM but no competitor. (b) % inhibition expressed as a function of inhibitor concentration. A total of 50% inhibition of unwinding of pDL3, four-way junction and G4 was achieved at 29.8, 2.73 and 29.88 nM, respectively. (c) Competition with cold four-way junction. (d) % inhibition expressed as a function of inhibitor concentration. A total of 50% inhibition of unwinding of the radiolabelled four-way junction substrate occurred at 1.91 nM. No significant inhibition of unwinding of either the plasmid-based D-loop or G4 DNA was found in the concentration range used. (e) Competition with cold G4 DNA. (f) % inhibition expressed as a function of inhibitor concentration. A total of 50% inhibition of unwinding of the pDL3 (blue triangle) and G4 DNA (red square) substrates occurred at 20.78 and 30.39 nM concentration, respectively.

Figure 6

Analysis of the recognition features important for efficient unwinding of plasmid-based D-loops. (a) Head-to-head comparison of the efficiency of unwinding of the different plasmid-based D-loops in a time-course study in the presence of 0.52 nM BLM. Samples were taken at the time points indicated above the lanes. (b) Quantification of data from (a). Symbols used were: red square, pDL5; green diamond, pDL55; blue double-triangle, pDLm; purple triangle, pDL3; cyan circle, pDL33.