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. 2000 Jun 6;97(12):6504-8.
doi: 10.1073/pnas.100448097.

The Bloom's syndrome gene product promotes branch migration of holliday junctions

J K Karow  1 , A Constantinou, J L Li, S C West, I D Hickson
Affiliations

The Bloom's syndrome gene product promotes branch migration of holliday junctions

J K Karow et al. Proc Natl Acad Sci U S A. .

Abstract

Bloom's syndrome (BS) is an autosomal recessive disorder associated with dwarfism, immunodeficiency, reduced fertility, and elevated levels of many types of cancer. BS cells show marked genomic instability; in particular, hyperrecombination between sister chromatids and homologous chromosomes. This instability is thought to result from defective processing of DNA replication intermediates. The gene mutated in BS, BLM, encodes a member of the RecQ family of DExH box DNA helicases, which also includes the Werner's syndrome gene product. We have investigated the mechanism by which BLM suppresses hyperrecombination. Here, we show that BLM selectively binds Holliday junctions in vitro and acts on recombination intermediates containing a Holliday junction to promote ATP-dependent branch migration. We present a model in which BLM disrupts potentially recombinogenic molecules that arise at sites of stalled replication forks. Our results have implications for the role of BLM as an anti-recombinase in the suppression of tumorigenesis.

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Figures

Figure 1
Figure 1
Branch migration of recombination intermediates catalyzed by BLM. (a) Schematic diagram indicating the formation of recombination intermediates (α-structures) by RecA and BLM-mediated branch migration back to the starting substrates. *, 32P labels. (b) Recombination intermediates were incubated with BLM, RuvAB, or PcrA as indicated, and the 32P-labeled products were analyzed as described in Methods. The linear duplex product of branch migration (L) is indicated. (c) Time course showing that BLM (20 nM) is unable to unwind a 488-bp linear duplex (1 nM).
Figure 2
Figure 2
Effect of ATP and RuvA on BLM-mediated branch migration. Reactions were carried out as described in Fig. 1. (a) Time courses were conducted in standard buffer (containing ATP), or in buffer from which ATP was omitted or replaced with 2 mM adenosine 5′-[β,γ-imido]triphosphate (AMP-PNP). (b) The formation of 32P-labeled branch migration products in a was quantified by phosphorimaging and expressed as a percentage of total radiolabel. (c) Recombination intermediates were preincubated with or without RuvA, as indicated, for 3 min on ice. BLM (18 nM) was then added, and reactions were incubated at 37°C for 45 min. Branch migration products were analyzed as in Fig. 1.
Figure 3
Figure 3
BLM binds selectively to synthetic X-junctions. Band-shift assays were carried out as described in Methods using 0.5 nM substrate representing either 32P-labeled X-junction (lanes a–e), 3′-tailed duplex DNA (lanes f–j), ssDNA (lanes k–o), or blunt-ended dsDNA (lanes p–t), together with the indicated concentrations of BLM.
Figure 4
Figure 4
Competition for binding of BLM to the X-junction. Band-shift assays were carried out using BLM (68 nM) and 32P-labeled X-junction (0.5 nM) as described in Methods. (a) Unlabeled X-junction competitor; (b) ssDNA competitor; (c) dsDNA competitor; (d) 3′-tailed duplex competitor; (e) RuvA competitor. Lanes: a, no competitor; b, 3.1 nM; c, 6.3 nM; d, 12.5 nM; e, 25 nM; f, 50 nM; g, 100 nM competitor in each case.
Figure 5
Figure 5
Model for the role of BLM as an anti-recombinase at sites of blocked replication forks. At sites of stalled forks (a), nascent DNA strands can dissociate from the template and anneal to each other, generating a Holliday junction (b). If this structure is not destroyed by the reverse branch migration activity of BLM, as indicated, junction resolution can occur (c) with the generation of a dsDNA break (d) and the subsequent formation of recombinants. See text for details.
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