Mammalian SMC3 C-terminal and Coiled-coil Protein Domains Specifically Bind Palindromic DNA, Do Not Block DNA Ends, and Prevent DNA Bending
1999; Elsevier BV; Volume: 274; Issue: 53 Linguagem: Inglês
10.1074/jbc.274.53.38216
ISSN1083-351X
AutoresAlexandre T. Akhmedov, Brigitte Gross, Rolf Jessberger,
Tópico(s)Genomics and Chromatin Dynamics
ResumoThe C-terminal domains of yeast structural maintenance of chromosomes (SMC) proteins were previously shown to bind double-stranded DNA, which generated the idea of the antiparallel SMC heterodimer, such as the SMC1/3 dimer, bridging two DNA molecules. Analysis of bovine SMC1 and SMC3 protein domains now reveals that not only the C-terminal domains, but also the coiled-coil region, binds DNA, while the N terminus is inactive. Duplex DNA and DNA molecules with secondary structures are highly preferred substrates for both the C-terminal and coiled-coil domains. Contrasting other cruciform DNA-binding proteins like HMG1, the SMC3 C-terminal and coiled-coil domains do not bend DNA, but rather prevent bending in ring closure assays. Phosphatase, exonuclease, and ligase assays showed that neither domain renders DNA ends inaccessible for other enzymes. These observations allow modifications of models for SMC-DNA interactions. The C-terminal domains of yeast structural maintenance of chromosomes (SMC) proteins were previously shown to bind double-stranded DNA, which generated the idea of the antiparallel SMC heterodimer, such as the SMC1/3 dimer, bridging two DNA molecules. Analysis of bovine SMC1 and SMC3 protein domains now reveals that not only the C-terminal domains, but also the coiled-coil region, binds DNA, while the N terminus is inactive. Duplex DNA and DNA molecules with secondary structures are highly preferred substrates for both the C-terminal and coiled-coil domains. Contrasting other cruciform DNA-binding proteins like HMG1, the SMC3 C-terminal and coiled-coil domains do not bend DNA, but rather prevent bending in ring closure assays. Phosphatase, exonuclease, and ligase assays showed that neither domain renders DNA ends inaccessible for other enzymes. These observations allow modifications of models for SMC-DNA interactions. structural maintenance of chromosomes bovine SMC yeast SMC dithiothreitol N-[2-hydroxyethyl]piperazine-N′3-propanesulfonic acid high mobility group 1 protein base pair(s) nucleotide(s) double-stranded DNA single-stranded DNA Structural maintenance of chromosomes (SMC)1 proteins are ubiquitous chromosomal components, evolutionary conserved from most prokaryotes to higher eukaryotes (for recent reviews, see Refs. 1Koshland D. Strunnikov A. Annu. Rev. Cell. Dev. Biol. 1996; 12: 305-333Crossref PubMed Scopus (286) Google Scholar, 2Heck M.M.S. Cell. 1997; 91: 5-8Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 3Jessberger R. Frei C. Gasser S.M. Curr. Opin. Genet. Dev. 1998; 8: 254-259Crossref PubMed Scopus (79) Google Scholar, 4Hirano T. Curr. Opin. Cell Biol. 1998; 10: 317-322Crossref PubMed Scopus (73) Google Scholar, 5Strunnikov A.V. Trends Cell Biol. 1998; 8: 454-459Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 6Hirano T. Genes Dev. 1999; 13: 11-19Crossref PubMed Scopus (198) Google Scholar, 7Strunnikov A.V. Jessberger R. Eur. J. Biochem. 1999; 263: 6-13Crossref PubMed Scopus (154) Google Scholar). The four characteristic members of the SMC protein family, generally termed SMC1 to SMC4, are involved in several aspects of chromosome dynamics. They form heterodimers, which are contained in higher order multiprotein complexes serving specific biological functions. In eukaryotes, so far two types of heterodimers have been observed, SMC1/3 and SMC2/4. In prokaryotic organisms, the SMC proteins form homodimers that are crucial for successful chromosome partition during cell division (8Melby T.E. Ciampaglio C.N. Briscoe G. Erickson H.P. J. Cell Biol. 1998; 142: 1595-1604Crossref PubMed Scopus (331) Google Scholar, 9Britton R.A. Lin D.C. Grossman A.D. Genes Dev. 1998; 12: 1254-1259Crossref PubMed Scopus (242) Google Scholar, 10Graumann P.L. Losick R. Strunnikov A.V. J. Bacteriol. 1998; 180: 5749-5755Crossref PubMed Google Scholar). So far, four different biological roles have been assigned to eukaryotic SMC protein complexes. The two most common are chromosome condensation (11Strunnikov A.V. Hogan E. Koshland D. Genes Dev. 1995; 9: 587-599Crossref PubMed Scopus (290) Google Scholar, 12Saka Y. Sutani T. Yamashita Y. Saitoh S. Takeuchi M. Nakaseko Y. Yanagida M. EMBO J. 1994; 13: 4938-4952Crossref PubMed Scopus (285) Google Scholar, 13Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar) and sister chromatid cohesion (14Guacci V. Koshland D. Strunnikov A. Cell. 1997; 91: 47-58Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar, 15Michaelis C. Ciosk R. Nasmyth K. Cell. 1997; 91: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1160) Google Scholar, 16Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (510) Google Scholar) during mitosis, exerted by complexes based on the SMC2/4 and the SMC1/3 heterodimers, respectively. In addition, SMC proteins function in sex chromosome gene dosage compensation in nematodes as an SMC2/4 heterodimer (17Chuang P.T. Lieb J.D. Meyer B. Science. 1996; 274: 1736-1738Crossref PubMed Scopus (81) Google Scholar, 18Lieb J.D. Albrecht M.R. Chuang P.T. Meyer B.J. Cell. 1998; 92: 1-20Abstract Full Text Full Text PDF Scopus (141) Google Scholar), and, with SMC1/3 as necessary subunits of the protein complex RC-1, act in DNA recombination and repair reactions (19Jessberger R. Riwar B. Baechtold H. Akhmedov A.T. EMBO J. 1996; 15: 4061-4068Crossref PubMed Scopus (112) Google Scholar). Smc3p has been recently noted to also be required for meiotic sister chromatid cohesion and reciprocal meiotic recombination in yeast (20Klein F. Mahr P. Galova M. Buonomo S.B.C. Michaelis C. Nairz K. Nasmyth K. Cell. 1999; 98: 91-103Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar).SMC proteins, with molecular masses between 110 and 170 kDa, share a unique structure that has been described in detail in a number of reviews (3Jessberger R. Frei C. Gasser S.M. Curr. Opin. Genet. Dev. 1998; 8: 254-259Crossref PubMed Scopus (79) Google Scholar, 4Hirano T. Curr. Opin. Cell Biol. 1998; 10: 317-322Crossref PubMed Scopus (73) Google Scholar, 5Strunnikov A.V. Trends Cell Biol. 1998; 8: 454-459Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 6Hirano T. Genes Dev. 1999; 13: 11-19Crossref PubMed Scopus (198) Google Scholar, 7Strunnikov A.V. Jessberger R. Eur. J. Biochem. 1999; 263: 6-13Crossref PubMed Scopus (154) Google Scholar). In brief, SMC proteins are characterized by two extended coiled-coil domains separated by a short hinge region of about 150 amino acids. The N- and C-terminal globular domains of about 100–150 amino acids are highly conserved and carry important motifs. The N-terminal domain contains an NTP binding motif (Walker A box; Ref.21Walker J.E. Sarasate M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4223) Google Scholar), which has been shown to bind the ATP analog azido-ATP (22Akhmedov A.T. Frei C. Tsai-Pflugfelder M. Kemper B. Gasser S.M. Jessberger R. J. Biol. Chem. 1998; 273: 24088-24094Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and the C-terminal domain contains a DA box (21Walker J.E. Sarasate M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4223) Google Scholar). While the N-terminal domain does not bind to DNA, the C-terminal domain does. TheSaccharomyces cerevisiae Smc1p and Smc2p C-terminal domains have been analyzed in some detail with respect to their interaction with DNA (22Akhmedov A.T. Frei C. Tsai-Pflugfelder M. Kemper B. Gasser S.M. Jessberger R. J. Biol. Chem. 1998; 273: 24088-24094Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). These domains show a strong preference of at least 100-fold for double-stranded DNA (dsDNA) substrates and a high specificity for such dsDNA molecules, which are able to adopt secondary structures. Synthetic cruciform DNA, as well as naturally occurring palindromic DNA sequences with the potential to form secondary structures, serve as the best substrates in gel shift experiments. Likewise, efficient competitors were scaffold-associated regions and budding yeast centromere DNA-derived fragments (22Akhmedov A.T. Frei C. Tsai-Pflugfelder M. Kemper B. Gasser S.M. Jessberger R. J. Biol. Chem. 1998; 273: 24088-24094Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Overall, there are good indications for a preference of yeast SMC C-terminal domains for specific DNA structures. Since studies on SMC binding to DNA are rather limited at present, many questions remain, for example whether the coiled-coil regions are directly involved in DNA binding and what effects SMC binding has on the substrate DNA.Electron microscopy studies of the Bacillus subtilis SMC homodimer revealed that the two SMC molecules probably form an antiparallel dimer, with colocalization of the C and N termini at each end (8Melby T.E. Ciampaglio C.N. Briscoe G. Erickson H.P. J. Cell Biol. 1998; 142: 1595-1604Crossref PubMed Scopus (331) Google Scholar). The most frequently observed shape of the homodimer is that of a completely folded rod, but SMC molecules can also exist as extended rods of about 100 nm in length and partially folded dimers. This indicates that they can move around the central hinge. Such a structure, upon formation of a complex between SMC dimer and DNA, might allow bipolar attachment and formation of a flexible protein bridge between two DNA molecules (6Hirano T. Genes Dev. 1999; 13: 11-19Crossref PubMed Scopus (198) Google Scholar, 7Strunnikov A.V. Jessberger R. Eur. J. Biochem. 1999; 263: 6-13Crossref PubMed Scopus (154) Google Scholar). Based on the high degree of evolutionary conservation, it is currently assumed, although not proven, that very similar structures are formed by the eukaryotic heterodimers.Key to all currently known biological activities of SMC proteins are interactions with DNA. Therefore, we set out to study DNA binding and bending by the terminal domains of mammalian (bovine) bSMC1 and bSMC3 proteins. Since mutant studies on Schizosaccharomyces pombeSMC4/2 protein homologs Cut3/Cut14 indicated that the second coiled-coil region may be involved in interactions with DNA (23Sutani T. Yanagida M. Nature. 1997; 388: 798-801Crossref PubMed Scopus (115) Google Scholar), we also included the corresponding bovine SMC3 coiled-coil region protein in the analysis.DISCUSSIONIn this paper on mammalian SMC protein domains, we report hitherto undescribed interactions with DNA: (i) the ability of the coiled-coil region alone to bind DNA, (ii) the strong preference of this interaction for duplex DNA and DNA substrates containing secondary structures, (iii) the inability of the coiled-coil and the C-terminal domains to bend DNA, but rather to inhibit bending, and (iv) the absence of a DNA binding activity that blocks DNA ends.An indication for an interaction of coiled-coil domains with DNA was revealed in mutant studies of S. pombe Cut3/Cut14 SMC proteins (SMC4/2 homologs). These proteins support reannealing of DNA complementary strands, but mutations within the coiled-coil regions of Cut3 (at amino acid 1147) and Cut14 (at amino acid 861) significantly reduced the activity of the heterodimer in that in vitroassay (23Sutani T. Yanagida M. Nature. 1997; 388: 798-801Crossref PubMed Scopus (115) Google Scholar). Our use of the bSMC1-C protein, which contains both the C-terminal domain and a large part of the adjacent coiled-coil region, indicated that the coiled-coil region at least does not inhibit DNA binding by the C terminus. Testing an isolated stretch of the bSMC3 coiled-coil region, which is relatively far apart from the C-terminal domain, showed that the coiled-coil region alone binds DNA and thus contributes to the DNA binding properties of full-length SMC3 protein. Binding of the yeast and bovine C-terminal and coiled-coil SMC proteins was observed with DNA substrates ranging from 139 to 422 bp in length. Double-stranded DNA served far better as a competitor than ssDNA. For their biological function in sister chromatid cohesion, binding of SMC1 and SMC3 proteins to newly synthesized daughter DNA duplexes, and not to single-stranded regions in DNA seems logical. Since none of the DNA binding activities of SMC protein domains investigated so far were unique for any particular type of SMC1, SMC2, or SMC3, one may speculate that the properties reported here are of a general nature for the eukaryotic SMC protein family. The high degree of conservation between eukaryotic SMC proteins supports such a hypothesis. Differences contributing to the distinct biological roles of the two SMC heterodimers may for example be reflected in hitherto unknown characteristics of the SMC proteins themselves, the heterodimers, or the holocomplexes.Proteins like those of the HMG family have been repeatedly shown to bind preferentially to secondary structured DNA, and to efficiently bend DNA in ring closure assays (28Kotlarz D. Fritsch A. Buc H. EMBO J. 1986; 5: 799-803Crossref PubMed Scopus (57) Google Scholar, 29Hodges-Garcia Y. Hagerman P.J. Pettijohn D.E. J. Biol. Chem. 1989; 264: 14621-14623Abstract Full Text PDF PubMed Google Scholar, 30Lyubchenko Y. Shlyakhtenko L. Chernov B. Harrington R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5331-5334Crossref PubMed Scopus (54) Google Scholar, 31Kahn J.D. Crothers D.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 89: 6343-6347Crossref Scopus (158) Google Scholar, 32Pil P.M. Chow C.S. Lippard S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9465-9469Crossref PubMed Scopus (195) Google Scholar, 33Paull T.T. Haykinson M.J. Johnson R.C. Genes Dev. 1993; 7: 1521-1534Crossref PubMed Scopus (309) Google Scholar, 34Stros M. J. Biol. Chem. 1998; 273: 10355-10361Abstract Full Text Full Text PDF PubMed Google Scholar, 35Giese K. Pagel J. Grosschedl R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12845-12850Crossref PubMed Scopus (57) Google Scholar). In contrast, the SMC protein domains, although displaying a very similar DNA substrate binding specificity, do not promote circularization of a 189- or 123-bp DNA fragment and thus appear not to bend DNA. Moreover, they prevent bending and may stabilize an extended, rod-like, linear structure of the DNA. Since such a negative result may have been caused by nonspecific factors, we included several controls, which showed that the ends of the linear DNA fragment are not blocked by the SMC proteins and are accessible to proteins and that the DNA ligase is active in the presence of the various SMC protein preparations. It may be noted that, in the mammalian multiprotein complex RC-1, the SMC1 and SMC3 proteins associate with a DNA ligase, which is also active in the presence of the two SMC proteins (38Jessberger R. Podust V. Hübscher U. Berg P. J. Biol. Chem. 1993; 268: 15070-15079Abstract Full Text PDF PubMed Google Scholar). The likely ability of SMC proteins to promote straightening of DNA seems to be evolutionary conserved, since the C-terminal and the coiled-coil region of bSMC3 and the S. cerevisiae SMC2-C protein behaved similarly. For the function of the SMC1/3 heterodimer in sister chromatid cohesion, such straightening of DNA may be advantageous for local linear juxtaposition of the two duplexes. Avoiding bends and kinks may also reduce steric hindrances not only for establishing sister chromatid cohesion but also in subsequent chromosome condensation steps. An overbent α-form with ends protruding into distance too far apart to be ligated may be an alternative structure generated in the ring closure assay. However, not only would this require a very high degree of bending especially of the 123-bp substrate, but also the known sequence-independent bending proteins are active in the ring closure assay regardless of their ability to overbend and form loops (39Zlatanova J. van Holde K. FASEB J. 1998; 12: 421-431Crossref PubMed Scopus (85) Google Scholar).The preference of SMC C-terminal and coiled-coil domains for DNA that has the potential to form secondary structures (i.e.palindromic DNA) or for synthetic cruciform DNA substrates apparently is also conserved from yeast (22Akhmedov A.T. Frei C. Tsai-Pflugfelder M. Kemper B. Gasser S.M. Jessberger R. J. Biol. Chem. 1998; 273: 24088-24094Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) to mammalian SMC proteins. Together, this specificity was observed for the C-terminal and coiled-coil domains of SMC1, SMC2, and SMC3 proteins, which at least in part represent the two biologically relevant heterodimers SMC1/3 and SMC2/4. It is remarkable that such differently structured domains as the globular C terminus and the coiled-coil domains display similar DNA interaction specificities. How can the preferential binding to secondary structures be reconciled with the anti-bending activity of the proteins? Removing bends and straightening DNA may happen to just such DNA secondary structures, as the equilibrium gets shifted toward the nonfolded, linear structure at a palindromic sequence. Thus, targeting the anti-bend protein SMC to secondary structures may further help in establishing a straight DNA configuration, more amenable to sister chromatin cohesion, chromosome condensation, and perhaps DNA recombination.As observed in the phosphatase, exonuclease, and ligase activity control assays in the bending experiments, the C-terminal and coiled-coil proteins do not interfere with the accessibility of dsDNA ends such as the 5′ protruding or blunt ends used in these assays. Thus, the SMC3 protein is not a DNA end-protecting protein. Limited protection of a stretch of internal DNA was observed in the exonuclease III experiments. This may be in agreement with the suggestion by Kleinet al. for yeast cohesins including Smc3p to protect meiotic double strand breaks from hyperresection (20Klein F. Mahr P. Galova M. Buonomo S.B.C. Michaelis C. Nairz K. Nasmyth K. Cell. 1999; 98: 91-103Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar). For their function in sister chromatid cohesion, which is established shortly after replicative synthesis of the two daughter duplexes, targeting to ends of a DNA duplex would have been unexpected, for there is normally no double strand break. This also implies that for DNA recombination reactions, such as strand invasion in sister chromatid exchange, or end-joining reactions mediated by DNA end-binding proteins like the DNA-dependent protein kinase holoenzyme, potentially recombinogenic DNA ends would probably not be blocked by the SMC3 protein.The DNA binding activities of the C-terminal yeast SMC proteins together with the antiparallel configuration of the SMC dimer, led to recent models, which invoke binding of both ends of an SMC heterodimer to two separate DNA molecules, thus in effect forming a bridge between them. Such models may help to explain the alignment of two duplex DNA molecules in postreplicative sister chromatid cohesion, which is co-mediated by the SMC1/3 heterodimer. An intramolecular DNA bridging model may likewise be important in chromosome condensation, facilitated by SMC2/4, since it possibly allows the connection of two regions many nanometers apart within one DNA duplex molecule and subsequently coiling them up. Connecting and perhaps even actively bringing two DNA duplexes together through the scissor-like movements around the central hinge of SMC proteins may also promote DNA repair and recombination reactions, e.g. at the pairing step.Our finding of a DNA binding capacity of the coiled-coil domain allows us to refine this model (Fig. 9). The extent of SMC protein binding to DNA is larger than previously thought, with possibly only the central hinge region positioned between (or above) the two DNA molecules. This arrangement may also better accommodate the dimensions of the molecules; while the two strands in a DNA duplex are about 2 nm apart from each other and the two duplexes in the aligned sister chromatid cohesion structure are within a distance of a few nanometers, the folded SMC heterodimer is 50 nm in length and, if extended, up to 100 nm (8Melby T.E. Ciampaglio C.N. Briscoe G. Erickson H.P. J. Cell Biol. 1998; 142: 1595-1604Crossref PubMed Scopus (331) Google Scholar). If bound only at its very tips to the two to-be-bridged DNA molecules, it probably would have to protrude considerably (Fig. 9 A). This dimensional problem may be solved by the view suggested here (Fig. 9 B). For DNA recombination and repair at double strand breaks, the extended binding of the two DNA duplexes may provide an advantageous structural setting, since it holds the two sister chromatids closely together and keeps the DNA ends at such a break in position (Fig. 9 C). This may support the initiation of recombination and repair of a double-strand break, which may have arisen during DNA replication or independent thereof (40Haber J.E. Trends Biochem. Sci. 1999; 24: 271-275Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). It remains possible, as has been proposed (19Jessberger R. Riwar B. Baechtold H. Akhmedov A.T. EMBO J. 1996; 15: 4061-4068Crossref PubMed Scopus (112) Google Scholar, 20Klein F. Mahr P. Galova M. Buonomo S.B.C. Michaelis C. Nairz K. Nasmyth K. Cell. 1999; 98: 91-103Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar), that the SMC proteins play both a structural and an active role in recombination and repair reactions. In any case, it will be important in future studies to determine how the DNA is bound and acted upon by full-length SMC proteins, the heterodimers, or holocomplexes. Structural maintenance of chromosomes (SMC)1 proteins are ubiquitous chromosomal components, evolutionary conserved from most prokaryotes to higher eukaryotes (for recent reviews, see Refs. 1Koshland D. Strunnikov A. Annu. Rev. Cell. Dev. Biol. 1996; 12: 305-333Crossref PubMed Scopus (286) Google Scholar, 2Heck M.M.S. Cell. 1997; 91: 5-8Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 3Jessberger R. Frei C. Gasser S.M. Curr. Opin. Genet. Dev. 1998; 8: 254-259Crossref PubMed Scopus (79) Google Scholar, 4Hirano T. Curr. Opin. Cell Biol. 1998; 10: 317-322Crossref PubMed Scopus (73) Google Scholar, 5Strunnikov A.V. Trends Cell Biol. 1998; 8: 454-459Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 6Hirano T. Genes Dev. 1999; 13: 11-19Crossref PubMed Scopus (198) Google Scholar, 7Strunnikov A.V. Jessberger R. Eur. J. Biochem. 1999; 263: 6-13Crossref PubMed Scopus (154) Google Scholar). The four characteristic members of the SMC protein family, generally termed SMC1 to SMC4, are involved in several aspects of chromosome dynamics. They form heterodimers, which are contained in higher order multiprotein complexes serving specific biological functions. In eukaryotes, so far two types of heterodimers have been observed, SMC1/3 and SMC2/4. In prokaryotic organisms, the SMC proteins form homodimers that are crucial for successful chromosome partition during cell division (8Melby T.E. Ciampaglio C.N. Briscoe G. Erickson H.P. J. Cell Biol. 1998; 142: 1595-1604Crossref PubMed Scopus (331) Google Scholar, 9Britton R.A. Lin D.C. Grossman A.D. Genes Dev. 1998; 12: 1254-1259Crossref PubMed Scopus (242) Google Scholar, 10Graumann P.L. Losick R. Strunnikov A.V. J. Bacteriol. 1998; 180: 5749-5755Crossref PubMed Google Scholar). So far, four different biological roles have been assigned to eukaryotic SMC protein complexes. The two most common are chromosome condensation (11Strunnikov A.V. Hogan E. Koshland D. Genes Dev. 1995; 9: 587-599Crossref PubMed Scopus (290) Google Scholar, 12Saka Y. Sutani T. Yamashita Y. Saitoh S. Takeuchi M. Nakaseko Y. Yanagida M. EMBO J. 1994; 13: 4938-4952Crossref PubMed Scopus (285) Google Scholar, 13Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar) and sister chromatid cohesion (14Guacci V. Koshland D. Strunnikov A. Cell. 1997; 91: 47-58Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar, 15Michaelis C. Ciosk R. Nasmyth K. Cell. 1997; 91: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1160) Google Scholar, 16Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (510) Google Scholar) during mitosis, exerted by complexes based on the SMC2/4 and the SMC1/3 heterodimers, respectively. In addition, SMC proteins function in sex chromosome gene dosage compensation in nematodes as an SMC2/4 heterodimer (17Chuang P.T. Lieb J.D. Meyer B. Science. 1996; 274: 1736-1738Crossref PubMed Scopus (81) Google Scholar, 18Lieb J.D. Albrecht M.R. Chuang P.T. Meyer B.J. Cell. 1998; 92: 1-20Abstract Full Text Full Text PDF Scopus (141) Google Scholar), and, with SMC1/3 as necessary subunits of the protein complex RC-1, act in DNA recombination and repair reactions (19Jessberger R. Riwar B. Baechtold H. Akhmedov A.T. EMBO J. 1996; 15: 4061-4068Crossref PubMed Scopus (112) Google Scholar). Smc3p has been recently noted to also be required for meiotic sister chromatid cohesion and reciprocal meiotic recombination in yeast (20Klein F. Mahr P. Galova M. Buonomo S.B.C. Michaelis C. Nairz K. Nasmyth K. Cell. 1999; 98: 91-103Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar). SMC proteins, with molecular masses between 110 and 170 kDa, share a unique structure that has been described in detail in a number of reviews (3Jessberger R. Frei C. Gasser S.M. Curr. Opin. Genet. Dev. 1998; 8: 254-259Crossref PubMed Scopus (79) Google Scholar, 4Hirano T. Curr. Opin. Cell Biol. 1998; 10: 317-322Crossref PubMed Scopus (73) Google Scholar, 5Strunnikov A.V. Trends Cell Biol. 1998; 8: 454-459Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 6Hirano T. Genes Dev. 1999; 13: 11-19Crossref PubMed Scopus (198) Google Scholar, 7Strunnikov A.V. Jessberger R. Eur. J. Biochem. 1999; 263: 6-13Crossref PubMed Scopus (154) Google Scholar). In brief, SMC proteins are characterized by two extended coiled-coil domains separated by a short hinge region of about 150 amino acids. The N- and C-terminal globular domains of about 100–150 amino acids are highly conserved and carry important motifs. The N-terminal domain contains an NTP binding motif (Walker A box; Ref.21Walker J.E. Sarasate M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4223) Google Scholar), which has been shown to bind the ATP analog azido-ATP (22Akhmedov A.T. Frei C. Tsai-Pflugfelder M. Kemper B. Gasser S.M. Jessberger R. J. Biol. Chem. 1998; 273: 24088-24094Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and the C-terminal domain contains a DA box (21Walker J.E. Sarasate M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4223) Google Scholar). While the N-terminal domain does not bind to DNA, the C-terminal domain does. TheSaccharomyces cerevisiae Smc1p and Smc2p C-terminal domains have been analyzed in some detail with respect to their interaction with DNA (22Akhmedov A.T. Frei C. Tsai-Pflugfelder M. Kemper B. Gasser S.M. Jessberger R. J. Biol. Chem. 1998; 273: 24088-24094Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). These domains show a strong preference of at least 100-fold for double-stranded DNA (dsDNA) substrates and a high specificity for such dsDNA molecules, which are able to adopt secondary structures. Synthetic cruciform DNA, as well as naturally occurring palindromic DNA sequences with the potential to form secondary structures, serve as the best substrates in gel shift experiments. Likewise, efficient competitors were scaffold-associated regions and budding yeast centromere DNA-derived fragments (22Akhmedov A.T. Frei C. Tsai-Pflugfelder M. Kemper B. Gasser S.M. Jessberger R. J. Biol. Chem. 1998; 273: 24088-24094Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Overall, there are good indications for a preference of yeast SMC C-terminal domains for specific DNA structures. Since studies on SMC binding to DNA are rather limited at present, many questions remain, for example whether the coiled-coil regions are directly involved in DNA binding and what effects SMC binding has on the substrate DNA. Electron microscopy studies of the Bacillus subtilis SMC homodimer revealed that the two SMC molecules probably form an antiparallel dimer, with colocalization of the C and N termini at each end (8Melby T.E. Ciampaglio C.N. Briscoe G. Erickson H.P. J. Cell Biol. 1998; 142: 1595-1604Crossref PubMed Scopus (331) Google Scholar). The most frequently observed shape of the homodimer is that of a completely folded rod, but SMC molecules can also exist as extended rods of about 100 nm in length and partially folded dimers. This indicates that they can move around the central hinge. Such a structure, upon formation of a complex between SMC dimer and DNA, might allow bipolar attachment and formation of a flexible protein bridge between two DNA molecules (6Hirano T. Genes Dev. 1999; 13: 11-19Crossref PubMed Scopus (198) Google Scholar, 7Strunnikov A.V. Jessberger R. Eur. J. Biochem. 1999; 263: 6-13Crossref PubMed Scopus (154) Google Scholar). Based on the high degree of evolutionary conservation, it is currently assumed, although not proven, that very similar structures are formed by the eukaryotic heterodimers. Key to all currently known biological activities of SMC proteins are interactions with DNA. Therefore, we set out to study DNA binding and bending by the terminal domains of mammalian (bovine) bSMC1 and bSMC3 proteins. Since mutant studies on Schizosaccharomyces pombeSMC4/2 protein homologs Cut3/Cut14 indicated that the second coiled-coil region may be involved in interactions with DNA (23Sutani T. Yanagida M. Nature. 1997; 388: 798-801Crossref PubMed Scopus (115) Google Scholar), we also included the corresponding bovine SMC3 coiled-coil region protein in th
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