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DNA branched junctions have been constructed that contain either five arms or six arms surrounding a branch point. These junctions are not as stable as junctions containing three or four arms; unlike the smaller junctions, they cannot be shown to migrate as a single band on native gels when each of their arms contains eight nucleotide pairs. However, they can be stabilized if their arms contain 16 nucleotide pairs. Ferguson analysis of these junctions in combination with three-arm and four-arm junctions indicates a linear increase in friction constant as the number of arms increases, with the four-arm junction migrating anomalously. The five-arm junction does not appear to have any unusual stacking structure, and all strands show similar responses to hydroxyl radical autofootprinting analysis. By contrast, one strand of the six-arm junction shows virtually no protection from hydroxyl radicals, suggesting that it is the helical strand of a preferred stacking domain. Both junctions are susceptible to digestion by T4 endonuclease VII, which resolves Holliday junctions. However, the putative helical strand of the six-arm junction shows markedly reduced cleavage, supporting the notion that its structure is largely found in a helical conformation. Branched DNA molecules can be assembled into structures whose helix axes form multiply connected objects and networks. The ability to construct five-arm and six-arm junctions vastly increases the number of structures and networks that can be built from branched DNA components. Icosahedral deltahedra and 11 networks with 432 symmetry, constructed from Platonic and Archimedean solids, are among the structures whose construction is feasible, now that these junctions can be made.

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An assay was developed that detected DNA cruciform cutting endonuclease activity in crude extracts of Saccharomyces cerevisiae. A collection of temperature‐sensitive strains was screened using this assay, and a mutant lacking the activity was found. The mutation leading to the enzymatic defect was mapped to the left arm of chromosome XI within 3 cM of the centromere. Cloning of the gene for this endonuclease was achieved by chromosome walking from the nearby PUT3 locus. The gene, called CCE1 (cruciform cutting endonuclease), was sequenced and found to have an open reading frame encoding a 41 kDa protein. The amino acid sequence of this eukaryotic endonuclease shows homology neither to its prokaryotic counterparts nor to other proteins in available databases. A cce1 null mutant has no obvious growth defect, and despite the ability of the CCE1 enzyme to cleave Holliday junction analogs, the mutant shows no defect in meiotic or mitotic recombination. A second cruciform cutting activity was detected in extracts from a cce1 null mutant, indicating that yeast has at least two such enzymes. The only phenotype observed for cce1 mutants is a higher than normal frequency of appearance of petite cells, suggesting that the CCE1 protein is important for the maintenance of mitochondrial DNA.

Structural maintenance of chromosomes (SMC) proteins interact with DNA in chromosome condensation, sister chromatid cohesion, DNA recombination, and gene dosage compensation. How individual SMC proteins and their functional domains bind DNA has not been described. We demonstrate the ability of the Cterminal domains of Saccharomyces cerevisiae SMC1 and SMC2 proteins, representing two major subfamilies with different functions, to bind DNA in an ATP-independent manner. Three levels of DNA binding specificity were observed: 1) a >100-fold preference for doublestranded versus single-stranded DNA; 2) a high affinity for DNA fragments able to form secondary structures and for synthetic cruciform DNA molecules; and 3) a strong preference for AT-rich DNA fragments of particular types. These include fragments from the scaffoldassociated regions, and an alternating poly(dA-dT)-poly(dT-dA) synthetic polymer, as opposed to a variety of other polymers. Reannealing of complementary DNA strands is also promoted primarily by the C-terminal domains. Consistent with their in vitro DNA binding activity, we show that overexpression of the SMC C termini increases plasmid loss without altering viability or cell cycle progression.The structural maintenance of chromosomes (SMC) 1 protein family, with members from lower and higher eukaryotes, may be divided into four subfamilies (SMC1 to SMC4) and two SMC-like protein subfamilies (SMC5 and SMC6) (for reviews see Refs. 1-7). Members of this family appear to function primarily as heterodimers and are implicated in a large range of activities that modulate chromosome structure and organization. Studies of yeast strains deficient in SMC1 and SMC2 show defects in the segregation of mitotic chromosomes (8 -10). A role in chromosome condensation was demonstrated in a cell-free chromosome condensation assay based on Xenopus laevis oocyte extracts (11), in which SMC2 and SMC4 subtypes were identified as essential components of the condensin protein complex (12). More recently, the Smc1p and Smc3p of Saccharomyces cerevisiae were shown to be essential for sister chromatid cohesion (13,14), and in Caenorhabditis elegans, two SMC protein homologs, MIX-1 and DPY-27, are involved in gene dosage compensation (15, 16). Finally, evidence for a role of SMC proteins in DNA recombination and repair has been supported by both genetic and biochemical studies. The Schizosaccharomyces pombe Rad18 gene and its S. cerevisiae homolog RHC18 are SMC-like and were found to act in an unusual postreplicative recombinational repair pathway (17), whereas mammalian SMC1 and SMC3 proteins were described as essential subunits of a recombinational repair protein complex (RC-1), isolated from calf thymus (18,19).SMC proteins display a very characteristic structure: two coiled-coil domains separate evolutionarily conserved head and tail domains, which contain an NTP binding motif (Walker A box) in the N terminus, and a DA box (Walker B) in the C terminus (20). The similarity of SMC proteins to motor proteins such as kinesin a...