DNA sliding clamps form an oligomeric ring encircling DNA and serve as a moving platform for DNA-processing proteins. The opening and closing of a sliding-clamp ring is essential to load the clamp onto DNA in order to perform its functions. The molecular details of how clamp rings open and enclose DNA are still not clear. Three PCNA homologues have been found in Sulfolobus solfataricus which form a heterotrimer. Taking advantage of their hetero-oligomeric nature, the structures of the PCNAs in monomeric PCNA3, dimeric PCNA1-PCNA2 and trimeric PCNA1-PCNA2-PCNA3 forms were determined at resolutions of 2.6-1.9 Å . The distinct oligomeric structures represent different stages in ring formation, which were verified in solution by ultracentrifugation analysis. The heterodimer opens in a V-shape of 130 , while the heterotrimers form a ring with a 120 rotation between monomers. The association of a rigid PCNA3 monomer with an opened PCNA1-PCNA2 heterodimer closes the ring and introduces a spring tension in the PCNA1-PCNA2 interface, thus bending the nine-stranded intermolecular -sheet to fit the 120 rotation. The release of the spring tension as PCNA3 dissociates from the ring may facilitate ring opening. The structural features in different assemblies present a molecular model for clamp ring assembly and opening. PDB References: PCNA3, 2ijx, r2ijxsf; PCNA1-PCNA2, 2io4, r2io4sf; PCNA1-PCNA2-PCNA3, 2nti, r2ntisf.

PCNA is a ring-shaped protein that encircles DNA and is essential for DNA metabolism, including DNA replication and repair. PCNA is either a homotrimer in eukaryotes and euryarchaeotes or a heterotrimer in some crenarchaeotes. The crenarchaeon Sulfolobus solfataricus encodes three PCNA homologues (PCNA1, PCNA2, and PCNA3). PCNA1 and PCNA2 form a stable dimer. The dimer then recruits PCNA3 to form the trimeric ring-shaped molecule that is typical for all PCNA proteins. We crystallized the PCNA3 monomer, the PCNA1-PCNA2 heterodimer, and the PCNA1-PCNA2-PCNA3 heterotrimer. The crystals diffract X-ray to 1.9, 2.6, and 2.5 Å resolutions, respectively. SAD phasing and molecular replacement solutions have confirmed that the crystals do contain the corresponding monomer, dimer, and trimer.

RNase Sa3 produced by Streptomyces aureofaciens strain CCM 3239 belongs to the T1 family of microbial ribonucleases. It is closely related both to RNase Sa, studied in detail earlier, and to RNase Sa2 produced by the same microorganism. The most important property of RNase Sa3 is the relatively high cytotoxic activity, which was not observed for RNase Sa and Sa2. Recombinant RNase Sa3 was overexpressed in Escherichia coli and puri®ed to high homogeneity. The hanging-drop vapour-diffusion method was used for crystallization. The two crystal forms are trigonal P3 1 21 and tetragonal P4 1 2 1 2, with unit-cell parameters a = b = 64.7, c = 69.6 A Ê , = 120 and a = b = 34.0, c = 147.2 A Ê , respectively. They diffract to 2.0 and to 1.7 A Ê resolution, respectively, using synchrotron radiation. The asymmetric units of crystal forms I and II contain one molecule of the enzyme, which corresponds to V M = 3.8 A Ê 3 Da À1 with a solvent content of 68% and V M = 1.9 A Ê 3 Da À1 with a solvent content of 37%, respectively.

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