DNA Interaction and Dimerization of Eukaryotic SMC Hinge Domains

Abstract

The eukaryotic SMC1/SMC3 heterodimer is essential for sister chromatid cohesion and acts in DNA repair and recombination. Dimerization depends on the central hinge domain present in all SMC proteins, which is flanked at each side by extended coiled-coil regions that terminate in specific globular domains. Here we report on DNA interactions of the eukaryotic, heterodimeric SMC1/SMC3 hinge regions, using the two known isoforms, SMC1␣/SMC3 and the meiotic SMC1␤/SMC3. Both dimers bind DNA with a preference for double-stranded DNA and DNA rich in potential secondary structures. Both dimers form large protein-DNA networks and promote reannealing of complementary DNA strands. DNA binding but not dimerization depends on approximately 20 amino acids of transitional sequence into the coiledcoil region. Replacement of three highly conserved glycine residues, thought to be required for dimerization, in one of the two hinge domains still allows formation of a stable dimer, but if two hinge domains are mutated dimerization fails. Single-mutant dimers bind DNA, but hinge monomers do not. Together, we show that eukaryotic hinge dimerization does not require conserved glycines in both hinge domains, that only the transition into the coiled-coil region rather than the entire coiledcoil region is necessary for DNA binding, and that dimerization is required but not sufficient for DNA binding of the eukaryotic hinge heterodimer.Eukaryotic SMC (structural maintenance of chromosomes) proteins are essential for sister chromatid cohesion and proper chromosome condensation and also act in response to DNA damage, in DNA recombination and repair, and in gene dosage compensation (for recent reviews see Refs. 1-6).SMC proteins, which are between 120 and 160 kDa in molecular mass, feature a design reminiscent of motor proteins. The C-terminal and N-terminal globular domains are separated by extended coiled-coil regions and a hinge domain at the center. Six members of the eukaryotic SMC protein family are known, named SMC1-SMC6 for most eukaryotic organisms. They form three types of heterodimers: SMC1/SMC3, SMC2/SMC4, and SMC5/SMC6. One heterodimer each constitutes a core component of a larger multiprotein complex that acts in one of the key functions described for SMC proteins. The best described complexes are cohesin (7-9), which is based on SMC1/SMC3, and condensin with its SMC2/SMC4 core (10, 11). The SMC5/SMC6 heterodimer forms a multiprotein complex that acts in postreplicative recombinational repair of DNA damage (12, 13). Also, there are several other complexes such as a mammalian complex called RC-1, which has been implicated in DNA recombination and repair reactions (14, 15), a DNA damage response-dependent complex that forms upon phosphorylation of SMC1 (16, 17), and a gene dosage compensation complex in Caenorhabditis elegans that contains SMC2 and SMC4 homologs and is responsible for maintaining the same level of X chromosome expression in XX and XO animals (18). In meiotic cells, there exist at least two, probably more, meiosis-s...

Characterization ofArabidopsis thalianaSMC1 and SMC3: evidence that AtSMC3 may function beyond chromosome cohesion

“…While DNA binding studies have shown that SMC proteins can directly bind to DNA (Akhmedov et al, 1998;Akhmedov et al, 1999;Chiu et al, 2004), similar studies have not been performed for the klesin subunits. Therefore, it is generally thought that the SMC proteins are responsible for cohesin complex binding to the chromosomes.…”

How might cohesin hold sister chromatids together?

“…Mutagenesis of glycine residues within Smc1 and Smc3 at sites predicted to lie at the interface between their hinge dimerization domains destroys their interaction in vitro. Because of the dimeric hinge's doughnut-shaped structure, there exist two independent interaction surfaces, and interestingly, mutation of both is required to abolish their interaction, at least in vitro (Chiu et al 2004). The effect of mutations that specifically disrupt the two interaction surfaces have not yet been investigated in vivo.…”

A Multi-Step Pathway for the Establishment of Sister Chromatid Cohesion

“…The joint requirement for these two domains is even more surprising given their apparent physical separation (;40 nm) based upon electron micrographs of cohesin [13]. Interestingly, in vitro analyses of bacterial Smc complexes have implicated the hinge domain in single-stranded and double-stranded DNA binding [37,44] and DNA-dependent stimulation of the ATPase activity of the head [44]. Thus, our in vivo study combined with these in vitro studies support DNA-dependent functional interactions between the opposite ends of Smc complexes.…”

“…Indeed, site-directed mutagenesis of conserved Smc1 residues in the Hinge, Walker A, Walker B, and Signature motifs demonstrated that Smc1 must bind Mcd1 and Smc3, as well as bind and hydrolyze ATP in order for cohesin to bind chromatin [7,21,22,37]. While informative, these mutational analyses of cohesin-chromatin association leave key questions unanswered.…”