Mammalian SMC3 C-terminal and Coiled-coil Protein Domains Specifically Bind Palindromic DNA, Do Not Block DNA Ends, and Prevent DNA Bending

Alexandre T Akhmedov,Brigitte Gross,Rolf Jessberger

doi:10.1074/jbc.274.53.38216

Abstract

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.

Highlights

Structural maintenance of chromosomes (SMC)1 proteins are ubiquitous chromosomal components, evolutionary conserved from most prokaryotes to higher eukaryotes
Direct comparison of the S. cerevisiae ySMC2-C domain and the bovine bSMC3-C in binding to a 230-bp doublestranded DNA (dsDNA) substrate derived from M13mp18 revealed similar binding efficiencies (Fig. 2)
In 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

Summary

Introduction

Structural maintenance of chromosomes (SMC) proteins are ubiquitous chromosomal components, evolutionary conserved from most prokaryotes to higher eukaryotes (for recent reviews, see Refs. 1–7). 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. The Saccharomyces cerevisiae Smc1p and Smc2p C-terminal domains have been analyzed in some detail with respect to their interaction with DNA [22] These domains show a strong preference of at least 100-fold for doublestranded DNA (dsDNA) substrates and a high specificity for such dsDNA molecules, which are able to adopt secondary structures. The most frequently observed shape of the homodimer is that of a completely folded rod, but SMC molecules can exist as extended rods of about 100 nm in length and partially folded dimers This indicates that they can move around the central hinge. Since mutant studies on Schizosaccharomyces pombe SMC4/2 protein homologs Cut3/Cut indicated that the second coiled-coil region may be involved in interactions with DNA [23], we in-

Methods

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Conclusion