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• W3116989724 abstract "The Bloom syndrome complex is a DNA damage repair machine. It consists of several protein components which are functional in isolation, but interdependent in cells for the maintenance of accurate homologous recombination. Mutations to any of the genes encoding these proteins cause numerous physical and developmental markers as well as phenotypes of genome instability, infertility, and cancer predisposition. Here we review the published structural and biochemical data on each of the components of the complex: the helicase BLM, the type IA topoisomerase TOP3A, and the OB-fold-containing RMI and RPA subunits. We describe how each component contributes to function, interacts with each other, and the DNA that it manipulates/repairs. The Bloom syndrome complex is a DNA damage repair machine. It consists of several protein components which are functional in isolation, but interdependent in cells for the maintenance of accurate homologous recombination. Mutations to any of the genes encoding these proteins cause numerous physical and developmental markers as well as phenotypes of genome instability, infertility, and cancer predisposition. Here we review the published structural and biochemical data on each of the components of the complex: the helicase BLM, the type IA topoisomerase TOP3A, and the OB-fold-containing RMI and RPA subunits. We describe how each component contributes to function, interacts with each other, and the DNA that it manipulates/repairs. Bloom syndrome (MIM 210900) is a rare autosomal recessive disorder characterized by growth deficiency, sun-sensitive skin, infertility, and predisposition to diabetes and cancer (Cunniff et al., 2017Cunniff C. Bassetti J.A. Ellis N.A. Bloom’s syndrome: clinical spectrum, molecular pathogenesis, and cancer predisposition.Mol. Syndromol. 2017; 8: 4-23Crossref PubMed Scopus (7) Google Scholar). Cancers arise at a median age of 25 years, often at multiple primary sites (German, 1997German J. Bloom’s syndrome. XX. The first 100 cancers.Cancer Genet. Cytogenet. 1997; 93: 100-106Abstract Full Text PDF PubMed Scopus (224) Google Scholar). More recently, it has been shown that heterozygote carriers of certain Bloom syndrome mutations are also cancer prone (Thompson et al., 2012Thompson E.R. Doyle M.A. Ryland G.L. Rowley S.M. Choong D.Y.H. Tothill R.W. Thorne H. Barnes D.R. Li J. Ellul J. et al.Exome sequencing identifies rare deleterious mutations in DNA repair genes FANCC and BLM as potential breast cancer susceptibility alleles.PLoS Genet. 2012; 8Crossref Scopus (128) Google Scholar). Cancer predisposition and other phenotypes were first linked to defective DNA repair when chromosome instability was observed in Bloom syndrome patient cells (German and Crippa, 1966German J. Crippa L.P. Chromosomal breakage in diploid cell lines from Bloom’s syndrome and Fanconi’s anemia.Ann. Genet. 1966; 9: 143-154Google Scholar). Animal models of Bloom syndrome have demonstrated the vital link between accumulation of DNA damage and the phenotypes that recapitulate those seen in humans with the disorder (Holloway et al., 2010Holloway J.K. Morelli M.A. Borst P.L. Cohen P.E. Mammalian BLM helicase is critical for integrating multiple pathways of meiotic recombination.J. Cell Biol. 2010; 188: 779-789Crossref PubMed Scopus (43) Google Scholar; Warren et al., 2010Warren M. Chung Y.J. Howat W.J. Harrison H. McGinnis R. Hao X. McCafferty J. Fredrickson T.N. Bradley A. Morse H.C. Irradiated Blm-deficient mice are a highly tumor prone model for analysis of a broad spectrum of hematologic malignancies.Leuk. Res. 2010; 34: 210-220Crossref PubMed Scopus (0) Google Scholar). As such, Bloom syndrome has informed us about the important role of DNA damage in both cancer initiation and its response to treatment. Individuals with Bloom syndrome or a Bloom syndrome-like syndrome have biallelic or homozygous mutations in one of four different genes: BLM, TOP3A, RMI1, or RMI2 (Ellis et al., 1995Ellis N.A. Lennon D.J. Proytcheva M. Alhadeff B. Henderson E.E. German J. Somatic intragenic recombination within the mutated locus BLM can correct the high sister-chromatid exchange phenotype of Bloom syndrome cells.Am. J. Hum. Genet. 1995; 57: 1019-1027PubMed Google Scholar; Hudson et al., 2016Hudson D.F. Amor D.J. Boys A. Butler K. Williams L. Zhang T. Kalitsis P. Loss of RMI2 increases genome instability and causes a Bloom-like syndrome.PLoS Genet. 2016; 12: 1-24Crossref Scopus (8) Google Scholar; Martin et al., 2018Martin C.A. Sarlós K. Logan C.V. Thakur R.S. Parry D.A. Bizard A.H. Leitch A. Cleal L. Ali N.S. Al-Owain M.A. et al.Mutations in TOP3A cause a Bloom syndrome-like disorder.Am. J. Hum. Genet. 2018; 103: 221-231Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Cells with mutations in any of these genes display increased chromosomal aberrations, including breaks, translocations, and elevated sister chromatid exchange (Wu et al., 2006Wu L. Bachrati C.Z. Ou J. Xu C. Yin J. Chang M. Wang W. Li L. Brown G.W. Hickson I.D. BLAP75/RMI1 promotes the BLM-dependent dissolution of homologous recombination intermediates.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4068-4073Crossref PubMed Scopus (205) Google Scholar). These observations underline the role of BLM, TOP3A, RMI1, and RMI2 in genomic stability and the suppression of DNA damage. In particular, the protein products of these genes assemble into a single protein complex called the Bloom syndrome complex, also known as the BLM dissolvasome or BTRR (BLM, TOP3A, RMI1, RMI2). BLM encodes the Bloom syndrome helicase (BLM). BLM is one of five human RecQ helicases, a family of DNA unwinding proteins that maintain genome stability across all kingdoms of life (Monnat, 2010Monnat R.J. Human RECQ helicases: roles in DNA metabolism, mutagenesis and cancer biology.Semin. Cancer Biol. 2010; 20: 329-339Crossref PubMed Scopus (89) Google Scholar). Like most helicases, BLM converts double-stranded DNA (dsDNA) to two separate single strands during various steps of DNA repair. TOP3A encodes DNA topoisomerase IIIα (TOP3A), a type IA topoisomerase that can act to release linked DNA molecules. RMI1 and RMI2 encode RecQ-mediated instability proteins 1 and 2 (RMI1 and RMI2), which contain oligonucleotide-binding (OB) domains, crucial for the formation and stability of the complex. There are numerous published discussions describing the biochemistry of the Bloom syndrome complex (Mankouri and Hickson, 2007Mankouri H.W. Hickson I.D. The RecQ helicase-topoisomerase III-Rmi1 complex: a DNA structure-specific “dissolvasome”?.Trends Biochem. Sci. 2007; 32: 538-546Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar; Manthei and Keck, 2013Manthei K.A. Keck J.L. The BLM dissolvasome in DNA replication and repair.Cell. Mol. Life Sci. 2013; 70: 4067-4084Crossref PubMed Scopus (53) Google Scholar); however, in this review, we will refocus these descriptions through the lens of structural data, as key structures have been made available since these previous reviews. We will describe the reactions catalyzed by members of the Bloom syndrome complex, discuss how the complex is assembled, and relate how interactions between subunits stimulate complex activity. A molecular view is also critical to our understanding of the obligate roles of Bloom syndrome complex in normal biological processes as diverse as meiosis (Holloway et al., 2010Holloway J.K. Morelli M.A. Borst P.L. Cohen P.E. Mammalian BLM helicase is critical for integrating multiple pathways of meiotic recombination.J. Cell Biol. 2010; 188: 779-789Crossref PubMed Scopus (43) Google Scholar), immunity (Gratia et al., 2019Gratia M. Rodero M.P. Conrad C. Samra E.B. Maurin M. Rice G.I. Duffy D. Revy P. Petit F. Dale R.C. et al.Bloom syndrome protein restrains innate immune sensing of micronuclei by cGAS.J. Exp. Med. 2019; 216: 1199-1213Crossref PubMed Scopus (18) Google Scholar), tumor suppression (Goss et al., 2002Goss K.H. Risinger M.A. Kordich J.J. Sanz M.H. Straughen J.E. Slovek L.E. Capobianco A.J. German J. Boivin G.P. Groden J. Enhanced tumor formation in mice heterozygous for Blm mutation.Science. 2002; 297: 2051-2053Crossref PubMed Scopus (0) Google Scholar), and chemotherapy sensitivity (Cunniff et al., 2017Cunniff C. Bassetti J.A. Ellis N.A. Bloom’s syndrome: clinical spectrum, molecular pathogenesis, and cancer predisposition.Mol. Syndromol. 2017; 8: 4-23Crossref PubMed Scopus (7) Google Scholar). The Bloom syndrome complex participates in a specific process of DNA repair, termed homologous recombination (HR). HR is required to repair double-strand breaks in DNA, and during the recovery of stalled or broken replication forks (Liu and West, 2004Liu Y. West S.C. Happy hollidays: 40th anniversary of the Holliday junction.Nat. Rev. Mol. Cell Biol. 2004; Crossref PubMed Scopus (106) Google Scholar). The process uses a strictly matched second copy of the damaged sequence as a repair template, so HR is limited to replicative (S) or post-replicative (G2) phases of the cell cycle where a sister chromatid is available. Mechanistically, HR can be divided into three stages: pre-synapsis, synapsis, and post synapsis (Figure 1). During pre-synapsis, the 3' strand of the broken DNA end is converted to single-stranded DNA (ssDNA) via the concerted exonuclease action of the MRE11, CtIP, EXO1, and DNA2 nucleases (Huertas, 2010Huertas P. DNA resection in eukaryotes: deciding how to fix the break.Nat. Struct. Mol. Biol. 2010; 17: 11-16Crossref PubMed Scopus (253) Google Scholar). This process is known as resection (Figure 1A). Resected DNA is then coated with a filament of RAD51, which is promoted in part by the action of the breast cancer tumor suppressor proteins BRCA1 and BRCA2 (Davies et al., 2001Davies A.A. Masson J. Mcilwraith M.J. Stasiak A.Z. Stasiak A. Venkitaraman A.R. West S.C. 1-s2 Role of BRCA2 in control of the RAD51 recombination and DNA repair protein.Mol. Cell. 2001; 7: 273-282Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar; Zhao et al., 2017Zhao W. Steinfeld J.B. Liang F. Chen X. Maranon D.G. Jian Ma C. Kwon Y. Rao T. Wang W. Sheng C. et al.BRCA1-BARD1 promotes RAD51-mediated homologous DNA pairing.Nature. 2017; 550: 360-365Crossref PubMed Scopus (95) Google Scholar). The RAD51 filament stimulates invasion of the resected DNA into a homologous template, creating a displacement loop (D-loop, Figure 1B). Several proteins are involved in stabilizing the D-loop in vivo to promote synapsis, including RAD54 and RAD51-paralogs (Crickard et al., 2020Crickard J.B. Moevus C.J. Kwon Y. Sung P. Greene E.C. Rad54 drives ATP hydrolysis-dependent DNA sequence alignment during homologous recombination.Cell. 2020; 181: 1380-1394.e18Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar; Kurumizaka et al., 2001Kurumizaka H. Ikawa S. Nakada M. Eda K. Kagawa W. Takata M. Takeda S. Yokoyama S. Shibata T. Homologous-pairing activity of the human DNA-repair proteins Xrcc3·Rad51C.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5538-5543Crossref PubMed Scopus (0) Google Scholar; McIlwraith and West, 2008McIlwraith M.J. West S.C. DNA repair synthesis facilitates RAD52-mediated second-end capture during DSB repair.Mol. Cell. 2008; 29: 510-516Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Post synapsis, DNA polymerases are engaged to extend the 3' end of the invaded, broken DNA strand (Figure 1C). Several different outcomes are possible. (1) Short regions of newly synthesized DNA can be displaced from the template strand and annealed then ligated to the resected DNA that comes from the other side of the double strand break (the second end), in a process of synthesis-dependent strand annealing. (2) The second end is instead annealed to the displaced strand of the D-loop, creating an additional substrate for polymerase action. Complete DNA synthesis leads to the formation of a structure called the double Holliday junction, which topologically links the two parental DNA molecules. Their separation requires structure-specific dsDNA nucleases (resolution) or branch migration and the cleavage and reannealing of a single strand (dissolution) (reviewed extensively in Liu and West, 2004Liu Y. West S.C. Happy hollidays: 40th anniversary of the Holliday junction.Nat. Rev. Mol. Cell Biol. 2004; Crossref PubMed Scopus (106) Google Scholar). (3) If the repaired end does not have a corresponding downstream sequence with which to anneal (for example, when the original break was located at a replication fork), DNA synthesis can continue indefinitely toward the next origin (or the end of the chromosome) in a process called break-induced replication (BIR) (Saini et al., 2013Saini N. Ramakrishnan S. Elango R. Ayyar S. Zhang Y. Deem A. Ira G. Haber J.E. Lobachev K.S. Malkova A. Migrating bubble during break-induced replication drives conservative DNA synthesis.Nature. 2013; 502: 389-392Crossref PubMed Scopus (198) Google Scholar). HR is typically an error-free repair process; however, its fidelity is dependent upon caveats: (1) selection of the correct DNA template during synapsis, (2) selection of the correct second end and, (3) absence of exchange of genetic information (crossing over). The Bloom syndrome complex plays major roles during pre-synapsis, synapsis, and post synapsis to ensure that these three caveats are met. These contributions of Bloom syndrome complex to the fidelity of HR, as determined by biochemical experiments, are outlined as insets in Figure 1. During pre-synapsis, the Bloom syndrome complex promotes resection, through a direct interaction with DNA2 and EXO1, to enlarge the size of the ssDNA:RAD51 filament available for homology search (Nimonkar et al., 2011Nimonkar A.V. Genschel J. Kinoshita E. Polaczek P. Campbell J.L. Wyman C. Modrich P. Kowalczykowski S.C. BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair.Genes Dev. 2011; 25: 350-362Crossref PubMed Scopus (414) Google Scholar). During synapsis, the Bloom syndrome complex can evict an invading ssDNA strand from a D-loop when the sequence being invaded does not fully match (Bachrati et al., 2006Bachrati C.Z. Borts R.H. Hickson I.D. Mobile D-loops are a preferred substrate for the Bloom’s syndrome helicase.Nucleic Acids Res. 2006; 34: 2269-2279Crossref PubMed Scopus (159) Google Scholar). In addition, the Bloom syndrome complex encourages synapsis by binding and stabilizing RAD51 when it is present within a fully homologous D-loop (Bugreev et al., 2009Bugreev D.V. Mazina O.M. Mazin A.V. Bloom syndrome helicase stimulates RAD51 DNA strand exchange activity through a novel mechanism.J. Biol. Chem. 2009; 284: 26349-26359Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Post synapsis, the Bloom syndrome complex can stimulate BIR in the absence of a second end, by promoting DNA polymerase delta extension of the D-loop (Selak et al., 2008Selak N. Bachrati C.Z. Shevelev I. Dietschy T. van Loon B. Jacob A. Hübscher U. Hoheisel J.D. Hickson I.D. Stagljar I. The Bloom’s syndrome helicase (BLM) interacts physically and functionally with p12, the smallest subunit of human DNA polymerase δ.Nucleic Acids Res. 2008; 36: 5166-5179Crossref PubMed Scopus (0) Google Scholar), or promote capture of the second end if one is available. Alternatively, the Bloom syndrome complex is the only enzyme complex capable of untangling the double Holliday junction structure without a risk of crossovers occurring (Wu and Hickson, 2003Wu L. Hickson I.O. The Bloom’s syndrome helicase suppresses crossing over during homologous recombination.Nature. 2003; Crossref Scopus (791) Google Scholar). So, what happens to HR when the Bloom syndrome complex is absent? HR occurs with reduced fidelity regulation, decreased resection, and increased crossing over, with the consequence being recombination-mediated insertions, deletions, and chromosome rearrangements. In meiosis, this leads to infertility (Holloway et al., 2010Holloway J.K. Morelli M.A. Borst P.L. Cohen P.E. Mammalian BLM helicase is critical for integrating multiple pathways of meiotic recombination.J. Cell Biol. 2010; 188: 779-789Crossref PubMed Scopus (43) Google Scholar); in immune cells, the elevated levels of incomplete recombination trigger release of immuno-stimulatory DNA causing autoimmunity (Gratia et al., 2019Gratia M. Rodero M.P. Conrad C. Samra E.B. Maurin M. Rice G.I. Duffy D. Revy P. Petit F. Dale R.C. et al.Bloom syndrome protein restrains innate immune sensing of micronuclei by cGAS.J. Exp. Med. 2019; 216: 1199-1213Crossref PubMed Scopus (18) Google Scholar); and in epithelial cells the genomic rearrangements drive cancer evolution (Goss et al., 2002Goss K.H. Risinger M.A. Kordich J.J. Sanz M.H. Straughen J.E. Slovek L.E. Capobianco A.J. German J. Boivin G.P. Groden J. Enhanced tumor formation in mice heterozygous for Blm mutation.Science. 2002; 297: 2051-2053Crossref PubMed Scopus (0) Google Scholar). Both in vitro and in vivo experiments have demonstrated that, when BLM is removed, DNA resection length is shorter at breaks (Gravel et al., 2008Gravel S. Chapman J.R. Magill C. Jackson S.P. DNA helicases Sgs1 and BLM promote DNA double-strand break resection.Genes Dev. 2008; 22: 2767-2772Crossref PubMed Scopus (411) Google Scholar; Nimonkar et al., 2011Nimonkar A.V. Genschel J. Kinoshita E. Polaczek P. Campbell J.L. Wyman C. Modrich P. Kowalczykowski S.C. BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair.Genes Dev. 2011; 25: 350-362Crossref PubMed Scopus (414) Google Scholar). This means that, in Bloom syndrome patients, synapse is more likely to occur with the wrong sequence (leading to translocation and rearrangement), BIR at single-end breaks is reduced (with particular effect on repeat instability), and nuclease-mediated crossovers are elevated (which promotes loss of heterozygosity). Fidelity regulation by the Bloom syndrome complex is therefore critical to all aspects of HR. The phenotypes of Bloom syndrome, due to misregulated HR, are overlapping between patients who have BLM, TOP3A, RMI1, or RMI2 deficiencies. Below we will discuss the structural properties of each protein product of these genes, and how it behaves as a component of the Bloom syndrome complex during the above reactions. More than 60 different Bloom syndrome-associated patient mutations have been identified within the BLM gene (German et al., 2007German J. Sanz M.M. Ciocci S. Ye T.Z. Ellis N.A. Syndrome-causing mutations of the BLM gene in persons in the Bloom’s syndrome registry.Hum. Mutat. 2007; 28: 743-753Crossref PubMed Scopus (113) Google Scholar). These include nonsense, missense, and exon-skipping mutations. All BLM patient nonsense mutations are deleterious. Even the least severely truncating patient nonsense mutation lacks the C-terminal nuclear localization sequence (Hayakawa et al., 2000Hayakawa S. Kaneko H. Fukao T. Kasahara K. Matsumoto T. Furuichi Y. Kondo N. Characterization of the nuclear localization signal in the DNA helicase responsible for Bloom syndrome.Int. J. Mol. Med. 2000; 5: 477-561PubMed Google Scholar; Kaneko et al., 1997Kaneko H. Orii K.O. Matsui E. Shimozawa N. Fukao T. Matsumoto T. Shimamoto A. Furuichi Y. Hayakawa S. Kasahara K. et al.BLM (the causative gene of Bloom syndrome) protein translocation into the nucleus by a nuclear localization signal.Biochem. Biophys. Res. Commun. 1997; 240: 348-353Crossref PubMed Scopus (48) Google Scholar), the C-terminal ssDNA annealing domain, and part of the helicase and RNaseD C-terminal (HRDC) domain, resulting in dysfunctional protein with improper localization. BLM is an SF2 helicase (Karow et al., 1997Karow J.K. Chakraverty R.K. Hickson I.D. The Bloom’s syndrome gene product is a 3’-5’ DNA helicase.J. Biol. Chem. 1997; 272: 30611-30614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), of which there are approximately 102 in humans (Fairman-Williams et al., 2010Fairman-Williams M.E. Guenther U.-P. Jankowsky E. SF1 and SF2 helicases: family matters.Curr. Opin. Struct. Biol. 2010; 20: 313-324Crossref PubMed Scopus (471) Google Scholar). It can be further classified into the RecQ subfamily, which contains five human proteins. The 1,417-amino-acid protein contains, in order, an N-terminal domain, a RecQ domain, a zinc finger domain, a winged helix domain, HRDC domain, and a C-terminal domain (Figures 2A and 2B ). While the N- and C-terminal domains are outside of the helicase core of the enzyme, they are essential for directing recruitment of other protein partners and for interacting with substrate DNA. The N terminus contains binding sites for replication protein A (RPA) (Doherty et al., 2005Doherty K.M. Sommers J.A. Gray M.D. Lee J.W. von Kobbe C. Thoma N.H. Kureekattil R.P. Kenny M.K. Brosh Jr., R.M. Physical and functional mapping of the replication protein a interaction domain of the Werner and Bloom syndrome helicases.J Biol Chem. 2005; 280: 29494-29505Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), DNA topoisomerase 2-binding protein 1 (TopBP1) (Sun et al., 2017Sun L. Huang Y. Edwards R.A. Yang S. Blackford A.N. Niedzwiedz W. Glover J.N.M. Structural insight into BLM recognition by TopBP1.Structure. 2017; 25: 1582-1588.e3Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar), and higher-order association with other BLM molecules (Beresten et al., 1999Beresten S.F. Stan R. Van Brabant A.J. Ye T. Naureckiene S. Ellis N.A. Purification of overexpressed hexahistidine-tagged BLM N431 as oligomeric complexes.Protein Expr. Purif. 1999; 17: 239-248Crossref PubMed Scopus (47) Google Scholar), while the C-terminal residues 1,290–1,350 contains a domain required for ssDNA annealing (Cheok et al., 2005Cheok C.F. Wu L. Garcia P.L. Janscak P. Hickson I.D. The Bloom’s syndrome helicase promotes the annealing of complementary single-stranded DNA.Nucleic Acids Res. 2005; 33: 3932-3941Crossref PubMed Scopus (0) Google Scholar). Both the N and C termini of BLM bind to RAD51 (Bergeron et al., 2011Bergeron K.L. Murphy E.L. Brown L.W. Almeida K.H. Critical interaction domains between bloom syndrome protein and RAD51.Protein J. 2011; 30: 1-8Crossref PubMed Scopus (5) Google Scholar). TopBP1, RPA, and RAD51 are all genome stability proteins and their interactions with BLM highlight the coordinated and multifaceted nature of genome maintenance. The RecQ domain is the engine of the BLM helicase and is made up of two RecA-like folds (D1 and D2), which cooperatively harness the energy of ATP binding and hydrolysis to translocate along DNA (Newman et al., 2015Newman J.A. Savitsky P. Allerston C.K. Bizard A.H. Özer Ö. Sarlós K. Liu Y. Pardon E. Steyaert J. Hickson I.D. et al.Crystal structure of the Bloom’s syndrome helicase indicates a role for the HRDC domain in conformational changes.Nucleic Acids Res. 2015; 43: 5221-5235Crossref PubMed Scopus (36) Google Scholar; Swan et al., 2014Swan M.K. Legris V. Tanner A. Reaper P.M. Vial S. Bordas R. Pollard J.R. Charlton P.A. Golec J.M.C. Bertrand J.A. Structure of human Bloom’s syndrome helicase in complex with ADP and duplex DNA.Acta Crystallogr. Sect. D Biol. Crystallogr. 2014; 70: 1465-1475Crossref PubMed Scopus (0) Google Scholar). The ATP-binding site is at the interface between the two folds. D1 contains most of the residues required for ATP binding, while D2 contains the “arginine finger,” a critical residue that correctly orients the gamma-phosphate of ATP for hydrolysis (Ren et al., 2007Ren H. Dou S.X. Rigolet P. Yang Y. Wang P.Y. Amor-Gueret M. Xi X.G. The arginine finger of the Bloom syndrome protein: its structural organization and its role in energy coupling.Nucleic Acids Res. 2007; 35: 6029-6041Crossref PubMed Scopus (0) Google Scholar) (Figure 2Ci). The mechanism by which ATP hydrolysis drives DNA translocation has been determined in detail by studying other related enzymes (Soultanas et al., 1999Soultanas P. Dillingham M.S. Velankar S.S. Wigley D.B. DNA binding mediates conformational changes and metal ion coordination in the active site of PcrA helicase.J. Mol. Biol. 1999; 290: 137-148Crossref PubMed Scopus (97) Google Scholar; Velankar et al., 1999Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism.Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Google Scholar; Yarranton and Gefter, 1979Yarranton G.T. Gefter M.L. Enzyme-catalyzed DNA unwinding: studies on Escherichia coli rep protein.Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1658-1662Crossref PubMed Google Scholar) and is analogous to the movement of an inchworm (Figure 2D). Briefly, in a non-ATP-bound state, one RecA-like fold (the leading domain) binds DNA tightly and the other RecA-like fold (the following domain) binds DNA weakly (Figure 2Di). ATP binding induces a conformational change, where the two domains are brought together and oriented appropriately for ATP hydrolysis (Figure 2Dii). This conformational change loosens the grip of the leading domain on DNA and tightens the grip of the following domain on the DNA in the direction that the enzyme moves along DNA; i.e., if the enzyme is a 3'-5' translocase, the following domain will bind DNA 5' of where it was initially weakly bound. Upon ATP hydrolysis, the gamma-phosphate is released, removing the tether between the two domains and resulting in their separation, restoring the initial conformation (Figure 2Diii). The position where the leading domain is bound after a single round of ATP hydrolysis is a single base pair step along the DNA (Patel and Donmez, 2006Patel S.S. Donmez I. Mechanisms of helicases.J. Biol. Chem. 2006; 281: 18265-18268Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Successive rounds of ATP hydrolysis result in the translocase moving many base pairs along DNA in a continuous fashion. The directionality (or polarity) of helicase action is determined by which of the two RecA-like folds act as the leading and following domains (Pugh et al., 2012Pugh R.A. Wu C.G. Spies M. Regulation of translocation polarity by helicase domain 1 in SF2B helicases.EMBO J. 2012; 31: 503-514Crossref PubMed Scopus (45) Google Scholar). For BLM, RecA2 acts as the first domain, and accordingly BLM translocates in a 3' to 5' direction along the DNA backbone. As with all helicases, the translocation domain acts cooperatively with other domains within the protein to force the DNA strands apart. Crystal structures of the BLM helicase in complex with DNA reveal how this is achieved (Newman et al., 2015Newman J.A. Savitsky P. Allerston C.K. Bizard A.H. Özer Ö. Sarlós K. Liu Y. Pardon E. Steyaert J. Hickson I.D. et al.Crystal structure of the Bloom’s syndrome helicase indicates a role for the HRDC domain in conformational changes.Nucleic Acids Res. 2015; 43: 5221-5235Crossref PubMed Scopus (36) Google Scholar; Swan et al., 2014Swan M.K. Legris V. Tanner A. Reaper P.M. Vial S. Bordas R. Pollard J.R. Charlton P.A. Golec J.M.C. Bertrand J.A. Structure of human Bloom’s syndrome helicase in complex with ADP and duplex DNA.Acta Crystallogr. Sect. D Biol. Crystallogr. 2014; 70: 1465-1475Crossref PubMed Scopus (0) Google Scholar). A winged helix and zinc finger domain are together responsible for binding to the DNA substrate at the interface between ssDNA- and dsDNA and peeling it apart, like a zipper on a zip (Newman et al., 2015Newman J.A. Savitsky P. Allerston C.K. Bizard A.H. Özer Ö. Sarlós K. Liu Y. Pardon E. Steyaert J. Hickson I.D. et al.Crystal structure of the Bloom’s syndrome helicase indicates a role for the HRDC domain in conformational changes.Nucleic Acids Res. 2015; 43: 5221-5235Crossref PubMed Scopus (36) Google Scholar; Swan et al., 2014Swan M.K. Legris V. Tanner A. Reaper P.M. Vial S. Bordas R. Pollard J.R. Charlton P.A. Golec J.M.C. Bertrand J.A. Structure of human Bloom’s syndrome helicase in complex with ADP and duplex DNA.Acta Crystallogr. Sect. D Biol. Crystallogr. 2014; 70: 1465-1475Crossref PubMed Scopus (0) Google Scholar). The strand-separating hairpin motif within the winged helix domain of BLM inserts between the two DNA strands and melts it from the complementary strand as the protein translocates (Newman et al., 2015Newman J.A. Savitsky P. Allerston C.K. Bizard A.H. Özer Ö. Sarlós K. Liu Y. Pardon E. Steyaert J. Hickson I.D. et al.Crystal structure of the Bloom’s syndrome helicase indicates a role for the HRDC domain in conformational changes.Nucleic Acids Res. 2015; 43: 5221-5235Crossref PubMed Scopus (36) Google Scholar; Swan et al., 2014Swan M.K. Legris V. Tanner A. Reaper P.M. Vial S. Bordas R. Pollard J.R. Charlton P.A. Golec J.M.C. Bertrand J.A. Structure of human Bloom’s syndrome helicase in complex with ADP and duplex DNA.Acta Crystallogr. Sect. D Biol. Crystallogr. 2014; 70: 1465-1475Crossref PubMed Scopus (0) Google Scholar). As BLM moves along the DNA substrate, the DNA bases that come in to contact with the strand-separating hairpin are flipped out of the duplex from their Watson-Crick base-paired conformation by rotation about the phosphodiester backbone (Figures 2B and 2D). This flipping mechanism is an essential component of BLM-mediated helicase activity, as BLM on its own cannot melt dsDNA substrates that contain a modified, rigid backbone that does not permit rotation of the base (Garcia et al., 2004Garcia P.L. Bradley G. Hayes C.J. Krintel S. Soultanas P. Janscak P. RPA alleviates the inhibitory effect of vinylphosphonate internucleotide linkages on DNA unwinding by BLM and WRN helicases.Nucleic Acids Res. 2004; 32: 3771-3778Crossref PubMed Scopus (20) Google Scholar). The helicase activity of BLM is essential for the majority of its cellular functions. Several mutants of BLM that lack helicase activity have been generated, and they share in c" @default.
• W3116989724 created "2021-01-05" @default.
• W3116989724 creator A5052682625 @default.
• W3116989724 creator A5062411430 @default.
• W3116989724 date "2021-02-01" @default.
• W3116989724 modified "2023-10-15" @default.
• W3116989724 title "A Structural Guide to the Bloom Syndrome Complex" @default.
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