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Inheritance of each chromosome depends upon its centromere. A histone H3 variant, CENP-A, is essential for epigenetically marking centromere location. We find that CENP-A is quantitatively retained at the centromere upon which it is initially assembled. CENP-C binds to CENP-A nucleosomes and is a prime candidate to stabilize centromeric chromatin. Using purified components, we find that CENP-C reshapes the octameric histone core of CENP-A nucleosomes, rigidifies both surface and internal nucleosome structure, and modulates terminal DNA to match the loose wrap that is found on native CENP-A nucleosomes at functional human centromeres. Thus, CENP-C affects nucleosome shape and dynamics in a manner analogous to allosteric regulation of enzymes. CENP-C depletion leads to rapid removal of CENP-A from centromeres, indicating their collaboration in maintaining centromere identity.

We have determined the X-ray crystal structures of two DNA Holliday junctions (HJs) bound by Cre recombinase. The HJ is a four-way branched structure that occurs as an intermediate in genetic recombination pathways, including site-specific recombination by the lambda-integrase family. Cre recombinase is an integrase family member that recombines 34 bp loxP sites in the absence of accessory proteins or auxiliary DNA sequences. The 2.7 A structure of Cre recombinase bound to an immobile HJ and the 2.5 A structure of Cre recombinase bound to a symmetric, nicked HJ reveal a nearly planar, twofold-symmetric DNA intermediate that shares features with both the stacked-X and the square conformations of the HJ that exist in the unbound state. The structures support a protein-mediated crossover isomerization of the junction that acts as the switch responsible for activation and deactivation of recombinase active sites. In this model, a subtle isomerization of the Cre recombinase-HJ quaternary structure dictates which strands are cleaved during resolution of the junction via a mechanism that involves neither branch migration nor helical restacking.

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Structural models of site-specific recombinases from the lambda integrase family of enzymes have in the last four years provided an important new perspective on the three-dimensional nature of the recombination pathway. Members of this family, which include the bacteriophage P1 Cre recombinase, bacteriophage lambda integrase, the yeast Flp recombinase, and the bacterial XerCD recombinases, exchange strands between DNA substrates in a stepwise process. One pair of strands is exchanged to form a Holliday junction intermediate, and the second pair of strands is exchanged during resolution of the junction to products. Crystal structures of reaction intermediates in the Cre-loxP site-specific recombination system, together with recent biochemical studies in the field, support a "strand swapping" model for recombination that does not require branch migration of the Holliday junction intermediate in order to test homology between recombining sites.

Cre recombinase catalyzes site-specific recombination between two 34-bp loxP sites in a variety of DNA substrates. At the start of the recombination pathway, the loxP sites are each bound by two recombinase molecules, and synapsis of the sites is mediated by Cre-Cre interactions. We describe the structures of synaptic complexes formed between a symmetrized loxP site and two Cre mutants that are defective in strand cleavage. The DNA in these complexes is bent sharply at a single base pair step at one end of the crossover region in a manner that is atypical of protein-induced DNA bends. A large negative roll (؊49°) and a positive tilt (16°) open the major groove toward the center of the synapse and compress the minor groove toward the protein-DNA interface. The bend direction of the site appears to determine which of the two DNA substrate strands will be cleaved and exchanged in the initial stages of the recombination pathway. These results provide a structural basis for the observation that exchange of DNA strands proceeds in a defined order in some tyrosine recombinase systems. The Cre-loxS synaptic complex structure supports a model in which synapsis of the loxP sites results in formation of a Holliday junction-like DNA architecture that is maintained through the initial cleavage and strand exchange steps in the site-specific recombination pathway.Most site-specific recombination systems in bacteria and yeast fall into one of two large families that carry out a similar repertoire of genetic rearrangements, including integration and excision of bacteriophage genomes into and out of host chromosomes, resolution of transposition intermediates, regulation of gene expression and plasmid copy number by inversion of DNA segments, and resolution of multimeric circular replicons generated by homologous recombination events (reviewed in refs. 1 and 2). The integrase family, also known as the ''tyrosine recombinases'' (3), includes over 100 members that share a conserved catalytic domain responsible for cleavage and ligation of the DNA substrates (4). The second large family of site-specific recombinases includes the resolvase enzymes from the ␥␦-and Tn3 transposons and the Gin and Hin invertases (1).The integrase family proteins carry out site-specific recombination by stepwise cleavage and exchange of each strand in the DNA substrates. Conserved tyrosine residues first cleave the scissile phosphates in sites recognized specifically by the recombinase proteins to form transient 3Ј-phosphotyrosine linkages and free 5Ј-hydroxyl groups (Fig. 1a). Intermolecular attack of the resulting phosphotyrosine linkages by 5Ј-hydroxyl groups results in the exchange of the first pair of DNA strands and the formation of a Holliday junction intermediate. The Holliday intermediate is then a substrate for a second round of cleavage and strand exchange steps that results in recombinant products. The active site chemistry of the cleavage and ligation steps is similar to that described recently for the eukaryotic topoisomerase Ib enzy...