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Crystal Structure of the Bloom's Syndrome Helicase Indicates a Role

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Crystal Structure of the Bloom's Syndrome Helicase Indicates a Role Nucleic Acids Research Advance Access published April 21, 2015 Nucleic Acids Research, 2015 1 doi: 10.1093/nar/gkv373 Crystal structure of the Bloom’s syndrome helicase indicates a role for the HRDC domain in conformational changes Joseph A. Newman1,†, Pavel Savitsky1,†, Charles K. Allerston1, Anna H. Bizard2, Ozg¨ un¨ Ozer¨ 2, Kata Sarlos´ 2, Ying Liu2, Els Pardon3,4, Jan Steyaert3,4, Ian D. Hickson2 and Opher Gileadi1,* 1Structural Genomics Consortium, University of Oxford, ORCRB, Roosevelt Drive, Oxford OX3 7DQ, UK, 2Center for Chromosome Stability and Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Panum Institute, Building 18.1, Blegdamsvej 3B, 2200 Copenhagen N, Denmark, 3Structural Biology Downloaded from Brussels, Vrije Universiteit Brussel, Pleinlaan 2 , 1050 Brussels, Belgium and 4Structural Biology Research Center, VIB, Brussel, Pleinlaan 2, 1050 Brussels, Belgium Received January 13, 2015; Revised March 27, 2015; Accepted April 03, 2015 http://nar.oxfordjournals.org/ ABSTRACT some instability, including chromatid gaps and breaks (4), various chromosome structural rearrangements (5)andan Bloom’s syndrome helicase (BLM) is a member of the increase in the number of sister chromatid exchange (SCE) RecQ family of DNA helicases, which play key roles in events, the latter serving as a distinguishing feature for the the maintenance of genome integrity in all organism clinical diagnosis of BS (6). groups. We describe crystal structures of the BLM Bloom’s syndrome helicase (BLM), like all RecQ-family helicase domain in complex with DNA and with an an- helicases, acts as a 3 to 5 helicase (7) on a wide variety tibody fragment, as well as SAXS and domain asso- of DNA substrates including forked duplexes, G quadru- at Chadwick & RAL Libraries on April 23, 2015 ciation studies in solution. We show an unexpected plexes, 4-way junctions (8) and displacement loops (D nucleotide-dependent interaction of the core heli- loops) (9). It forms a multi protein complex with hu- ␣ case domain with the conserved, poorly character- man topoisomerase III (10), RMI1 (11) and RMI2 (12), ized HRDC domain. The BLM–DNA complex shows termed the ‘dissolvasome’, which can promote the con- vergent branch migration (13) and decatenation of dou- an unusual base-flipping mechanism with unique ble Holliday junction intermediates formed during homolo- positioning of the DNA duplex relative to the heli- gous recombination. This dissolution reaction prevents the case core domains. Comparison with other crystal exchange of genetic material flanking two homologous se- structures of RecQ helicases permits the definition quences engaged in homologous recombination (14,15). of structural transitions underlying ATP-driven heli- The BLM protein is a 1417 amino acid, 159-kDa case action, and the identification of a nucleotide- polypeptide with multiple structural domains (Figure 1A). regulated tunnel that may play a role in interactions At its centre (residues 639–1290) is a catalytic core con- with complex DNA substrates. served amongst RecQ-family helicases. A large N-terminal domain (residues 1–638) is believed to play a role in regu- lation and oligomerization of BLM (16,17), and is impor- INTRODUCTION tant in mediating interactions with partner proteins (10,18– Bloom’s syndrome (BS) is an extremely rare autosomal re- 19). The C-terminal region (residues 1291–1417), also in- cessive disorder that is characterized by shortness of stature, volved in protein interactions (10), is believed to be pre- a distinctive skin rash and the predisposition to the devel- dominantly unstructured, and contains a nuclear localiza- opment of a wide spectrum of cancers at an early age (1). tion signal (20). We have focussed this study on the core do- Individuals with BS carry mutations in BLM that result ei- main, which is responsible for the basic functions of DNA ther in expression of truncated proteins or alterations of key unwinding. conserved residues within the RecQ helicase domain (1–3). The core domain itself is composed of subdomains, with On a cellular level, BS is characterized by excessive chromo- varying degrees of evolutionary conservation (Figure 1A). *To whom correspondence should be addressed. Tel: +44 1865 617572; Fax: +44 1865 617575; Email: [email protected] †These authors contributed equally to the paper as first authors. Present address: Charles K. Allerston, Dart Neuroscience LLC San Diego, CA 92121, USA. C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2 Nucleic Acids Research, 2015 Downloaded from http://nar.oxfordjournals.org/ at Chadwick & RAL Libraries on April 23, 2015 Figure 1. Structure of the BLM Nanobody complex. (A) Conserved domains in BLM protein. (B) Structure of the BLM nanobody complex with domains labelled and coloured as in (A), and the ADP and bound zinc ion are shown in the sphere representation. This figure and all subsequent molecular graphics figures were created using the program PyMOL odinger(Schr¨ LCC). The adenosine triphosphate (ATP)-dependent motor ac- double Holliday junctions efficiently (28), suggesting that tivity resides in two RecA-like domains termed D1 and the HRDC domain plays a crucial role during dissolution. D2 (residues 639–857 and 858–993, respectively), which The structures of various HRDC domains have been de- contain sequence motifs shared amongst superfamily 2 termined in isolation (30–32). Despite the core structure (SF2) helicases (reviewed in (21)). Further downstream is being conserved, both the primary sequence and surface the RecQ family-specific C-terminal domain (RQC) that properties of the HRDC domains vary markedly, with the includes a helical hairpin and Zn2+ binding subdomain BLM HRDC surface being predominantly electronegative (residues 994–1068, termed Zn) and a Winged Helix (WH) whilst the HRDC domain from WRN is predominantly DNA binding domain (residues 1069–1192). Two of the ␤- electropositive (33). On the basis of these differences it has strands in the WH domain form a prominent hairpin that been suggested that the HRDC domain may play a role in has been suggested to act as a DNA strand separation pin in modulating interactions with other components in protein other RecQ helicases (22–24). Finally, residues 1193–1290 complexes (32). On the other hand, it has been suggested constitute the HRDC (helicase and RNase D C-terminal) that the HRDC domain binds to DNA (25). Indeed, numer- domain, which occurs in two of the five human RecQ-family ous studies have implicated various HRDC domains with helicases (BLM and WRN), as well as in RecQ helicases a direct role in DNA binding (30,31), although only very in bacteria and yeast (25); as its name suggests, it is also weak signle-stranded DNA (ssDNA) binding (Kd ∼ 100 present in other nucleic acid modifying enzymes including ␮M) could be detected in one study for the BLM HRDC RNase D and RNA polymerase II subunit 4 (25,26). BLM domain (34) whilst another similar study was unable to de- mutants lacking the HRDC domain, whilst possessing core tect any binding when testing at concentrations up to 10 ␮M helicase and adenosine triphosphatase (ATPase) activities (33). Hence, the mechanism underlying HRDC function in similar to wild-type protein (27–29), are defective in both dissolution remains obscure. strand annealing (27) and double Holliday junction dis- Determining the ways in which the structural domains solution (28). Moreover, a K1270V point mutation in the of BLM relate and interact with each other will be the key HRDC domain was found to affect the higher order func- to understanding the role of BLM in the maintenance of tions of BLM, giving rise to a protein unable to dissolve genome stability and how it is able to catalyze complex re- Nucleic Acids Research, 2015 3 actions such as double Holliday junction dissolution. In and incubation continued overnight at 180 RPM. The cells this study we present the 2.8-A˚ resolution crystal structure were collected by centrifugation and suspended in 100- of BLM636–1298, in complex with both adenosine diphos- ml HES buffer (200-mM 4-(2-Hydroxyethyl)piperazine-1- phate (ADP) and a single domain llama antibody fragment ethanesulfonic acid (HEPES), pH 7.5, 0.5-M sucrose, 0.5- (nanobody) specifically raised to BLM as an aid to crystal- mM ethylenediaminetetraacetic acid (EDTA)) and frozen lization. We also present two structures of BLM in complex at −80◦C in 50-ml aliquots. Fifty millilitre of cell suspen- with DNA at 4.7–3.5-A˚ resolution containing 12-bp duplex sion was thawed and kept on ice for 1 h with occasional DNA with single-stranded 3-overhangs. Comparisons to mixing. The cells were diluted with 50 ml of water. After other RecQ helicase structures reveals the highly dynamic another 1-h incubation on ice, the cells were removed by nature of the protein. Finally, we show that the HRDC do- centrifugation and the protein was recovered from the su- main packs tightly against the cleft formed between the two pernatant (periplasmic extract). The protein was then pu- RecA-like domains D1 and D2 and that this binding is sen- rified by binding to Ni-IDA Sepharose, which was washed sitive to the nucleotide status of the enzyme. and eluted with 0.3-M imidazole then further purified by gel filtration in Superdex S75 in 50-mM HEPES, pH 7.5, 0.5-M MATERIALS AND METHODS NaCl, 5% glycerol. 636–1298 BLM (spanning the helicase, RQC and HRDC Downloaded from Generation and selection of nanobodies domains) was expressed in E. coli and purified by Ni- Nanobody CA5075 resulted from a feasibility study for gen- Sepharose, tag cleavage, heparin-Sepharose and Superdex erating and selecting nanobodies against a large number of 200 as described in the Supplementary information of (36).
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