The HRDC domain of BLM is required for the dissolution of double Holliday junctions
2005; Springer Nature; Volume: 24; Issue: 14 Linguagem: Inglês
10.1038/sj.emboj.7600740
ISSN1460-2075
AutoresLeonard Wu, Kok‐Lung Chan, Christine Ralf, Douglas A. Bernstein, Patrick L. Garcia, Vilhelm A. Bohr, Alessandro Vindigni, Pavel Janščák, James L. Keck, Ian D. Hickson,
Tópico(s)Genetic Neurodegenerative Diseases
ResumoArticle30 June 2005free access The HRDC domain of BLM is required for the dissolution of double Holliday junctions Leonard Wu Leonard Wu Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK Search for more papers by this author Kok Lung Chan Kok Lung Chan Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK Search for more papers by this author Christine Ralf Christine Ralf Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK Search for more papers by this author Douglas A Bernstein Douglas A Bernstein Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA Search for more papers by this author Patrick L Garcia Patrick L Garcia Institute of Molecular Cancer Research, University of Zürich, Zürich, Switzerland Search for more papers by this author Vilhelm A Bohr Vilhelm A Bohr Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, MD, USA Search for more papers by this author Alessandro Vindigni Alessandro Vindigni International Centre for Genetic Engineering and Biotechnology, Padriciano, Trieste, Italy Search for more papers by this author Pavel Janscak Pavel Janscak Institute of Molecular Cancer Research, University of Zürich, Zürich, Switzerland Search for more papers by this author James L Keck James L Keck Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA Search for more papers by this author Ian D Hickson Corresponding Author Ian D Hickson Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK Search for more papers by this author Leonard Wu Leonard Wu Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK Search for more papers by this author Kok Lung Chan Kok Lung Chan Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK Search for more papers by this author Christine Ralf Christine Ralf Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK Search for more papers by this author Douglas A Bernstein Douglas A Bernstein Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA Search for more papers by this author Patrick L Garcia Patrick L Garcia Institute of Molecular Cancer Research, University of Zürich, Zürich, Switzerland Search for more papers by this author Vilhelm A Bohr Vilhelm A Bohr Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, MD, USA Search for more papers by this author Alessandro Vindigni Alessandro Vindigni International Centre for Genetic Engineering and Biotechnology, Padriciano, Trieste, Italy Search for more papers by this author Pavel Janscak Pavel Janscak Institute of Molecular Cancer Research, University of Zürich, Zürich, Switzerland Search for more papers by this author James L Keck James L Keck Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA Search for more papers by this author Ian D Hickson Corresponding Author Ian D Hickson Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK Search for more papers by this author Author Information Leonard Wu1, Kok Lung Chan1, Christine Ralf1, Douglas A Bernstein2, Patrick L Garcia3, Vilhelm A Bohr4, Alessandro Vindigni5, Pavel Janscak3, James L Keck2 and Ian D Hickson 1 1Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK 2Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA 3Institute of Molecular Cancer Research, University of Zürich, Zürich, Switzerland 4Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, MD, USA 5International Centre for Genetic Engineering and Biotechnology, Padriciano, Trieste, Italy *Corresponding author. Cancer Research UK Laboratories, Oxford Cancer Centre, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, UK. Tel.: +44 1865 222 417; Fax: +44 1865 222 431; E-mail: [email protected] The EMBO Journal (2005)24:2679-2687https://doi.org/10.1038/sj.emboj.7600740 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Bloom's syndrome is a hereditary cancer-predisposition disorder resulting from mutations in the BLM gene. In humans, BLM encodes one of five members of the RecQ helicase family. One function of BLM is to act in concert with topoisomerase IIIα (TOPO IIIα) to resolve recombination intermediates containing double Holliday junctions by a process called double Holliday junction dissolution, herein termed dissolution. Here, we show that dissolution is highly specific for BLM among human RecQ helicases and critically depends upon a functional HRDC domain in BLM. We show that the HRDC domain confers DNA structure specificity, and is required for the efficient binding to and unwinding of double Holliday junctions, but not for the unwinding of a simple partial duplex substrate. Furthermore, we show that lysine-1270 of BLM, which resides in the HRDC domain and is predicted to play a role in mediating interactions with DNA, is required for efficient dissolution. Introduction Mutations in the BLM gene give rise to Bloom's syndrome (BS) in humans (Ellis et al, 1995). This rare genetic disorder is characterized by short stature, sunlight sensitivity, immunodeficiency and a predisposition to the development of many different types of malignancies (Bachrati and Hickson, 2003; Hickson, 2003). BLM encodes a DNA helicase that belongs to a highly conserved family, the prototypical member of which is encoded by the recQ gene of Escherichia coli (Figure 1A) (Bachrati and Hickson, 2003). Germline mutations in two other human RecQ family helicase genes also give rise to cancer-predisposition disorders: WRN mutations give rise to Werner's syndrome and RECQ4 mutations are associated with some cases of Rothmund–Thomson syndrome (Yu et al, 1996; Kitao et al, 1999). Figure 1.(A) Schematic representation of selected members of the RecQ helicase family. All proteins are from humans, except E. coli RecQ and S. cerevisiae Sgs1, and are aligned about the conserved helicase domain. Also shown are the relative positions of the exonuclease (Exo) and ssDNA annealing domains of WRN and RECQ5β, respectively, and the RQC (R) and HRDC domains. Nonconserved regions are shaded in gray. Relevant amino-acid residue positions of truncation mutants used in this study are indicated above BLM and RecQ. See text for details. (B) Alignment of the HRDC domain of BLM, WRN, Sgs1 and E. coli RecQ. Residues comprising α-helices in the three-dimensional structure of Sgs1 are indicated by boxes above. Identical or similar residues are shaded in gray. Shown below is the amino-acid substitution K1270V used in the generation of BLM(K1270V). See text for details. The figure was adapted from Liu et al (1999). Download figure Download PowerPoint The existence of clinically distinct disorders associated with inactivation of any of three different RecQ family helicases indicates that the human RecQ family helicases have specialized functions in the cell (Oakley and Hickson, 2002; Bachrati and Hickson, 2003; Wang et al, 2003). In the budding yeast, Saccharomyces cerevisiae, mutation of the sole RecQ helicase gene, SGS1, results in elevated levels of genome-wide genetic recombination (Gangloff et al, 1994; Watt et al, 1996). This role in regulating homologous recombination levels appears to have been conserved in humans, at least in the case of BLM, since BS cells also show excessive levels of genome-wide homologous recombination. This is particularly apparent for recombination events between sister chromatids, giving rise to a characteristically elevated level of sister chromatid exchanges (SCEs) that is sufficiently specific for BS to be used in the diagnosis of the disorder (Chaganti et al, 1974). The hyper-recombination phenotype of certain RecQ helicase mutants is likely due to aberrant processing of recombination intermediates (Wu and Hickson, 2002b) since RecQ helicases from several organisms have been purified, and have been shown to act upon DNA intermediates that arise during the process of homologous recombination (Bachrati and Hickson, 2003). RecQ helicases all contain a conserved helicase domain that defines the family (Bachrati and Hickson, 2003). There are, however, additional sequence motifs that flank the signature helicase domain (Figure 1A). In certain cases, additional enzymatic activities reside in these flanking domains. In the case of WRN, a 3′–5′ exonuclease domain is located in the N-terminal region, whereas the C-terminal domain of RECQ5β directs a DNA strand annealing function (Huang et al, 1998; Kamath-Loeb et al, 1998; Garcia et al, 2004) (Figure 1A). Other sequence motifs identified in RecQ helicases include the RQC domain, which is situated C-terminal to the helicase domain and appears to be found exclusively in some RecQ helicases, and the HRDC domain, which is found C-terminal to the RQC domain of some RecQ helicases and in RNaseD homologs (Figure 1A and B) (Bachrati and Hickson, 2003). The structures of the RQC and HRDC domains have been determined for RecQ and Sgs1, respectively (Liu et al, 1999; Bernstein et al, 2003). The RQC domain resembles a so-called winged-helix domain, which is a member of the helix–turn–helix superfamily, and plays a role in binding duplex DNA (Bernstein et al, 2003). Interestingly, in BLM and WRN, the RQC domain has been implicated also in the orchestration of protein–protein interactions (Brosh et al, 2001; von Kobbe et al, 2002; Bachrati and Hickson, 2003). The HRDC domain is thought to function as an auxiliary DNA-binding domain and, structurally, it resembles domains found in many DNA-metabolizing enzymes such as helicases, polymerases and recombinases (Morozov et al, 1997; Liu et al, 1999). How these conserved motifs impact on the biochemical properties of RecQ helicases and affect the in vivo function of these enzymes has not been determined. In addition to the presence or absence of the aforementioned functional domains, the different cellular functions of the various vertebrate RecQ helicases likely depend on their ability to form specific interactions with different protein partners. BLM is proposed to act in a complex with replication protein A and the type IA topoisomerase, topoisomerase IIIα (hTOPO IIIα) (Brosh et al, 2000; Johnson et al, 2000; Wu et al, 2000; Meetei et al, 2003). This association with a type IA topoisomerase is evolutionarily conserved, since Sgs1p also acts together with Top3p, the only type IA topoisomerase expressed in budding yeast (Gangloff et al, 1994; Bennett and Wang, 2001; Fricke et al, 2001). top3 mutant cells have elevated levels of recombination (Wallis et al, 1989), which can be suppressed, at least partially, by inactivation of either Sgs1p or Rad52p, the latter of which is required for all forms of homologous recombination (Gangloff et al, 1994; Oakley et al, 2002; Shor et al, 2002). The conserved coupling of a RecQ helicase and a type IA topoisomerase, therefore, appears to have a central role in regulating recombination levels in the cell (Wu and Hickson, 2001). In particular, Sgs1p and BLM appear to suppress the formation of crossover products that arise from the resolution of homologous recombination intermediates containing Holliday junctions (Ira et al, 2003; Wu and Hickson, 2003). We have recently proposed a mechanism by which this is achieved by BLM. Together with hTOPO IIIα, BLM can catalyze double Holliday junction dissolution (DHJ), a reaction mechanism in which DHJs are resolved exclusively into non-crossover recombinant products (Wu and Hickson, 2003). Here, we have demonstrated that BLM cannot by substituted by other RecQ helicases in dissolution reactions containing hTOPO IIIα. Furthermore, we reveal the first biochemical function for the HRDC domain of a RecQ helicase through the demonstration of a requirement for this domain in the specific recognition, processing and dissolution of DHJs. Results Double Holliday junction dissolution is specific for BLM Despite the similar biochemical properties of many RecQ helicases in vitro, and their ability to act upon a wide variety of DNA structures, an elevated frequency of SCEs is thought to be a feature unique to BLM-deficient cells among human RecQ helicase mutants. We set out, therefore, to examine the apparent contradiction between, on the one hand, the promiscuous and redundant biochemical activities of RecQ helicases in vitro and, on the other, the distinct cellular phenotypes of RecQ helicase mutants. To do this, we analyzed the ability of three other human RecQ helicases to catalyze dissolution in conjunction with hTOPO IIIα. Dissolution was assessed using an oligonucleotide-based molecule (DHJ) that we had used previously to assay for dissolution activity (Figure 2) (Fu et al, 1994; Wu and Hickson, 2003). Using this substrate, we found that WRN, RECQ1 and RECQ5β could not substitute for BLM in dissolution reactions with hTOPO IIIα, indicating that the reaction mechanism of dissolution displays a high degree of specificity for BLM (Figure 3). Figure 2.Outline of the strategy used in the generation of the DHJ-containing substrates analyzed in this study, DHJ and DHJ/Rsa1. DHJ comprises two oligonucleotides (B1 and R1), each of which contains two regions of complementary sequences giving rise to two 11 bp internal duplex arms. When incubated together, B1 and R1 anneal to form a molecule that contains two juxtaposed HJs separated by two 14 bp heteroduplex regions (Fu et al, 1994; Wu and Hickson, 2003). These heteroduplexes contain approximately 1.5 helical turns, which results in the catenation of B1 and R1 when both oligonucleotides are circularized by ligation. Topologically relevant helical turns are shown. Each oligonucleotide can be detected individually by the 5′ end-labeling of one of the oligonucleotides prior to annealing. In this scheme, oligonucleotide R1 has been labeled, as indicated by the asterisk. Blue arrows indicate the enzymatic activity each substrate was used to assess and the detectable product generated from each substrate. Dissolution of DHJ results in the decatenation of B1 and R1 releasing the two oligonucleotides as circular DNA species, each of which has a faster mobility on denaturing PAGE than the DHJ substrate (Wu and Hickson, 2003). Unwinding of DHJ/Rsa1 by BLM releases linear R1 and circular B1. Download figure Download PowerPoint Figure 3.Dissolution is specifically catalyzed by BLM. Dissolution reactions containing a series of two-fold dilutions of BLM (upper panel, 5, 2.5, 1.25, 0.625, 0.3125, 0.15 and 0.075 nM; the middle and lower panels did not contain the three and two lowest concentrations, respectively), WRN (15, 7.5, 3.75, 1.87, 0.94, 0.47 and 0.24 nM), RECQ1 (100, 50, 25 and 12.5 nM) or RECQ5β (80, 40, 20, 10 and 5 nM), as indicated above the panels. Lanes containing hTOPO IIIα (250 nM) are indicated above by black bars. Positions of the DHJ substrate and the dissolution product are shown on the left. For simplicity, the intertwining of the strands of DHJ is omitted. Download figure Download PowerPoint The N-terminal domain of BLM is not required to catalyze dissolution The apparently specific ability of BLM to catalyze dissolution indicated that the BLM protein itself must possess biochemical functions that are not present in the other human RecQ helicases. To identify structural features of BLM that are required for catalysis of dissolution, we generated a series of truncated versions of BLM that all retained the catalytic core of BLM (Figure 1) and assessed the ability of these BLM variants to catalyze dissolution. Residues 1–133 of BLM contain an hTOPO IIIα-interaction domain (Hu et al, 2001). We therefore wanted to determine if this interaction domain was required for dissolution. Deletion of residues 1–212 of BLM, and thus removal of this hTOPO IIIα-interaction domain, did not affect the ability of BLM to catalyze dissolution (Figure 4A). Moreover, a truncated BLM protein generated by removal of the entire nonconserved N-terminal domain of BLM (residues 1–641) was still able to catalyze dissolution (data not shown). Dissolution of DHJ is a symmetrical reaction and generates two circular products in an ATP-dependent manner. To eliminate the possibility that both of the N-terminally truncated proteins catalyze a pseudodissolution reaction by, for instance, asymmetric nicking of the substrate in the unlabeled oligonucleotide, we monitored the fate of both oligonucleotides by performing dissolution reactions on the two forms of DHJ in which either oligonculeotide R1 or B1 was labeled. The reaction catalyzed by both of the N-terminal BLM truncation mutants on each DHJ substrate was indistinguishable from that carried out by full-length BLM with regard to ATP dependency and the structure of the reaction products, that is, two intact circular species (Figure 4A and B and data not shown). Together, these data indicate that the N-terminal domain of BLM, and thus a physical association of hTOPO IIIα with this portion of BLM, is not required for the catalysis of dissolution. Figure 4.The C- but not the N-terminal domain of BLM is required for dissolution. (A) Dissolution reactions in the presence or absence of 5 mM ATP and containing hTOPO IIIα and full-length BLM(1–1417) or BLM(213–1417), as indicated. In the DHJ substrate, oligonucleotide R1 is labeled. Positions of the DHJ substrate, dissolution product and products of the indicated restriction digests, confirming the identity of the labeled oligonucleotide, are shown on the left. (B) Identical reactions to those shown in (A) were carried out except that a DHJ molecule was used in which oligonucleotide B1 was labeled. (C) Dissolution reactions containing a series of two-fold dilutions of BLM (highest concentration, 5 nM) or BLM(213–1267) (highest concentration, 7 nM). Lanes containing hTOPO IIIα (250 nM) are indicated by black bars. Positions of the DHJ substrate and dissolution product are shown on the left. Download figure Download PowerPoint The C-terminal domain of BLM is essential for dissolution The dispensable nature of the N-terminal domain of BLM for dissolution led us to examine the role of the C-terminal domain of BLM using a previously described, truncated version of BLM that consists of residues 213–1267, herein designated BLM(213–1267) (Wu and Hickson, 2002a). Despite being an active helicase, we found that BLM(213–1267), in contrast to BLM(213–1417), which contains the natural C-terminus of BLM, was defective in catalyzing dissolution (Figure 4C). This indicates that in addition to the helicase domain, residues 1268–1417 of BLM are required for dissolution (Figure 4). The RecQ family HRDC domain is required for the binding to and unwinding of double Holliday junctions The deletion of residues 1268–1417 to generate BLM(213–1267) resulted in truncation of the HRDC domain, which consists of residues 1210–1290 in BLM (Figure 1A and B). We reasoned, therefore, that the failure of BLM(213–1267) to catalyze dissolution might be reflected in a diminished ability to interact with and/or process DHJs as a result of the inactivation of the DNA-binding function of the BLM HRDC domain. To address this, we compared the ability of BLM and BLM(213–1267) to unwind, in the absence of hTOPO IIIα, a modified DHJ molecule (DHJ/Rsa1) in which DHJ had been digested with Rsa1 (Wu and Hickson, 2003). The linearization of oligonucleotide R1 by Rsa1 results in the topological unlinking of the two constituent oligonucleotides of DHJ and thus creates a substrate that can be disrupted by thermal denaturation or by a helicase activity (Figure 2) (Wu and Hickson, 2003). The intact DHJ substrate is resistant to conventional unwinding due to its covalently closed nature. While we found that both BLM and BLM(213–1267) were able to unwind DHJ/Rsa1, BLM(213–1267) displayed an approximately 10-fold reduction in helicase activity toward this substrate compared to that of full-length BLM (Figure 5). This impaired helicase activity was solely due to the loss of residues 1268–1417 of BLM, since BLM(213–1417), which lacks only the N-terminal 1–212 residues, displayed helicase activity on this substrate that was equivalent to that displayed by full-length BLM (Figure 5). Figure 5.The C-terminal domain of BLM is required for efficient unwinding of DHJs. Helicase assays using DHJ/Rsa1 and a series of two-fold dilutions of BLM, BLM(213–1417) or BLM(213–1267) on DHJ/Rsa1, as indicated. The positions of DHJ/Rsa1 and the unwound product are shown on the right. The graph shows quantification of the level of unwound product generated by BLM (circles), BLM(213–1417) (squares) and BLM(213–1267) (triangles) on DHJ/Rsa1. Note that the scale on the horizontal axis is logarithmic. Download figure Download PowerPoint One possible explanation for the reduced helicase activity displayed by BLM(213–1267) was that the truncated protein has a reduced affinity for DHJs. To analyze this, we employed two independent means to compare the binding affinities of BLM(213–1417) and BLM(213–1267) for the DHJ substrate: a filter-binding assay, in which we measured protein-dependent retention of DHJ on a nitrocellulose membrane, and an electrophoretic mobility shift assay (EMSA), in which potentially different protein/DHJ complexes were visualized as a result of their reduced electrophoretic mobility compared to protein-free DHJ. Using the filter-binding assay, we found that BLM(213–1267) had a significantly reduced ability to bind DHJ as compared to BLM(213–1417) (Figure 6A). Using EMSAs, BLM(213–1417) was found to generate two differentially retarded species and a small amount of material that could not be resolved and was retained in the gel wells (Figure 6B). The level of all retarded species was dramatically reduced when BLM(213–1267) replaced BLM(213–1417) in the EMSA analyses (Figure 6B), thus confirming the conclusion of the filter-binding assay. Figure 6.The C-terminal domain of BLM is required for efficient binding to DHJs. (A) DNA filter-binding assay using a series of two-fold dilutions of BLM(213–1417) or BLM(213–1267) on DHJ. The graph shows the quantification of the relative level of DNA bound to the filter in reactions containing either BLM(213–1417) (squares) or BLM(213–1267) (triangles). (B) DNA EMSA using a series of two-fold dilutions of BLM(213–1417) or BLM(213–1267) on DHJ, as indicated above the lanes. The positions of the unbound substrate and protein–DNA complexes (black arrowheads) are indicated on the right. The graph shows the quantification of the total amount of DNA bound by BLM(213–1417) (squares) or BLM(213–1267) (triangles). Download figure Download PowerPoint Next, we addressed the question of whether the observed reduction in DNA-binding affinity and helicase activity of BLM(213–1267) was specific for DHJ structures. To do this, we compared the helicase activities of BLM, BLM(213–1417) and BLM(213–1267) on a conventional helicase substrate, a forked partial duplex. Interestingly, all three proteins displayed very similar levels of helicase activity on this substrate and, in contrast to the results with the DHJ substrate, BLM and BLM(213–1267) displayed similar binding affinities for the forked duplex substrate (Figure 7). Together, these data strongly suggest that the C-terminal domain of BLM contains a DNA structure-specific binding motif, and implicate residues 1268–1417 of BLM in the processing of DHJ structures. Figure 7.The C-terminal domain of BLM is not required for the unwinding or binding of forked duplexes. (A) Helicase assays using a series of two-fold dilutions of BLM, BLM(213–1417) and BLM(213–1267) on a forked partial duplex substrate. The positions of the forked duplex and the unwound product are shown on the right. The graph shows quantification of the amount of unwound product generated by BLM (circles), BLM(213–1417) (squares) or BLM(213–1267) (triangles). (B) DNA filter-binding assay using a series of two-fold dilutions of BLM or BLM(213–1267) on a forked partial duplex substrate. The graph shows quantification of the relative amount of DNA bound to the filter in reactions containing BLM(213–1417) (squares) or BLM(213–1267) (triangles). Download figure Download PowerPoint To provide further support for the proposal that it is loss of the HRDC domain that is responsible for the reduced ability of BLM(213–1267) to unwind DHJ/Rsa1, a comparison was made between the activities of E. coli RecQ and a truncated version of this protein (residues 1–523) that lacks the entire HRDC domain, RecQ(1–523) (Figure 1A and B) (Bernstein and Keck, 2003). Both proteins were able to unwind DHJ/Rsa1 to some extent, but RecQ(1–523) had a much reduced ability to unwind this molecule compared to full-length RecQ (>10-fold reduction; Figure 8). As was the case with BLM, loss of the RecQ HRDC domain appeared to specifically affect the helicase activity of RecQ on the DHJ/Rsa1 substrate since it had no significant effect on its helicase activity on a forked DNA substrate (Figure 8). These data suggest a conserved function for the HRDC domain of RecQ family helicases in the specific processing of DHJs. The fact that the RecQ and BLM HRDC domains apparently have similar functions in facilitating the efficient unwinding of DHJ/Rsa1 raised the possibility that RecQ might also catalyze dissolution together with bacterial Top3. RecQ and Top3 have been shown to cooperate in the mediation of a strand passage activity that facilitates the catenation and decatenation of covalently closed plasmid DNAs (Harmon et al, 1999). However, we found that RecQ and Top3 could not catalyze dissolution under conditions that supported dissolution by BLM and hTOPO IIIα (data not shown). Figure 8.The E. coli RecQ HRDC domain is required for the unwinding of DHJ/Rsa1 but not of a forked duplex. Helicase assays using RecQ and RecQ(1–523) on either DHJ/Rsa1 (left) or a forked partial duplex (right) substrate. The graphs show the quantification of unwound product for each substrate for RecQ (circles) and RecQ(1–523) (squares). Note that the scale on the horizontal axis is logarithmic. Download figure Download PowerPoint Lysine-1270 of BLM is required for efficient dissolution activity Thus far, we conclude that the inability of BLM(213–1267) to catalyze dissolution is likely due to the absence of a functional HRDC domain, which had been inactivated by truncation. However, to demonstrate this directly and to eliminate the possibility that the truncation of the C-terminal 150 residues to generate BLM(213–1267) might have had long-range effects on other portions of the BLM protein and thereby led to inactivation of non-HRDC-mediated functions, we sought to abrogate the HRDC domain function in the context of the full-length protein. The similarity of the predicted secondary structure of the HRDC domain in RecQ helicases to motifs in unrelated DNA-metabolizing enzymes suggests that it is the conserved protein fold of the HRDC domain that is important for function. We set out, therefore, specifically to disrupt the conformation of the HRDC domain in BLM by site-directed mutagenesis. Initial attempts involved either the substitution of proline for the highly conserved alanine-1250 residue in order to destabilize α-helix 2 of the HRDC domain, or the creation of an internal deletion removing residues 1251–1262 to truncate α-helix 2 and remove α-helix 3 (Figure 1B). However, neither of these proteins could be expressed successfully, indicating that disruption of the fold of the HRDC domain likely reduces the solubility and/or stability of BLM. As an alternative, we chose to mutate highly conserved residues that, in the predicted three-dimensional structure of the BLM HRDC domain, reside on the protein surface. We reasoned that such residues were less likely to be essential for the overall structural integrity of the HRDC domain, but may be important for function through the mediation of intermolecular interactions. Lysine-1329 of Sgs1 has been shown to contact ssDNA, consistent with the role of the HRDC domain as an auxiliary DNA-binding domain (Liu et al, 1999). This residue is conserved among RecQ helicases and corresponds to residue 1270 in BLM (Figure 1B). We therefore mutated the BLM cDNA to convert lysine-1270 to valine in the full-length BLM protein to generate BLM(K1270V). BLM(K1270V) could be expressed and purified to give protein yields that were comparable to those seen with wild-type BLM (data not shown). Purified BLM(K1270V) was active as a helicase, and had a level of activity on a forked partial duplex substrate that was indistinguishable from that of wild-type BLM (Figure 9A). The substitution of lysine-1270 to valine, therefore, did not appear to have any gross effect on the overall stability or the helicase activ
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