The crystal structures of two ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I (Klentaq1) with a primer/template DNA and dideoxycytidine triphosphate, and that of a binary complex of the same enzyme with a primer/template DNA, were determined to a resolution of 2.3, 2.3 and 2.5 Å, respectively. One ternary complex structure differs markedly from the two other structures by a large reorientation of the tip of the fingers domain. This structure, designated 'closed', represents the ternary polymerase complex caught in the act of incorporating a nucleotide. In the two other structures, the tip of the fingers domain is rotated outward by 46°('open') in an orientation similar to that of the apo form of Klentaq1. These structures provide the first direct evidence in DNA polymerase I enzymes of a large conformational change responsible for assembling an active ternary complex.

Crystal structures of binary and ternary complexes of the E. coli Rep helicase bound to single-stranded (ss) DNA or ssDNA and ADP were determined to a resolution of 3.0 A and 3.2 A, respectively. The asymmetric unit in the crystals contains two Rep monomers differing from each other by a large reorientation of one of the domains, corresponding to a swiveling of 130 degrees about a hinge region. Such domain movements are sufficiently large to suggest that these may be coupled to translocation of the Rep dimer along DNA. The ssDNA binding site involves the helicase motifs Ia, III, and V, whereas the ADP binding site involves helicase motifs I and IV. Residues in motifs II and VI may function to transduce the allosteric effects of nucleotides on DNA binding. These structures represent the first view of a DNA helicase bound to DNA.

Mutations in the cohesin regulators NIPBL and ESCO2 are causative of the Cornelia de Lange syndrome (CdLS) and Roberts or SC phocomelia syndrome, respectively. Recently, mutations in the cohesin complex structural component SMC1A have been identified in two probands with features of CdLS. Here, we report the identification of a mutation in the gene encoding the complementary subunit of the cohesin heterodimer, SMC3, and 14 additional SMC1A mutations. All mutations are predicted to retain an open reading frame, and no truncating mutations were identified. Structural analysis of the mutant SMC3 and SMC1A proteins indicate that all are likely to produce functional cohesin complexes, but we posit that they may alter their chromosome binding dynamics. Our data indicate that SMC3 and SMC1A mutations (1) contribute to approximately 5% of cases of CdLS, (2) result in a consistently mild phenotype with absence of major structural anomalies typically associated with CdLS, and (3) in some instances, result in a phenotype that approaches that of apparently nonsyndromic mental retardation.

The human single-stranded DNA-binding protein, replication protein A (RPA) binds DNA in at least two different modes: initial [8±10 nucleotides (nt)] and stable (~30 nt). Switching from 8 to 30 nt mode is associated with a large conformational change. Here we report the 2.8 A Ê structure of the RPA trimerization core comprising the C-terminal DNA-binding domain of subunit RPA70 (DBD-C), the central DNA-binding domain of subunit RPA32 (DBD-D) and the entire RPA14 subunit. All three domains are built around a central oligonucleotide/oligosaccharide binding (OB)-fold and¯anked by a helix at the C-terminus. Trimerization is mediated by three C-terminal helices arranged in parallel. The OB-fold of DBD-C possesses unique structural features; embedded zinc ribbon and helix±turn±helix motifs. Using time-resolved proteolysis with trypsin, we demonstrate that the trimerization core does not contribute to the binding with substrates of 10 nt, but interacts with oligonucleotides of 24 nt. Taken together, our data indicate that switching from 8±10 to 30 nt mode is mediated by DNA binding with the trimerization core.

Proliferating cell nuclear antigen (PCNA) protein is one of the central molecules responsible for decisions of life and death of the cell. The PCNA gene is induced by p53, while PCNA protein interacts with p53-controlled proteins Gadd45, MyD118, CR6 and, most importantly, p21, in the process of deciding cell fate. If PCNA protein is present in abundance in the cell in the absence of p53, DNA replication occurs. On the other hand, if PCNA protein levels are high in the cell in the presence of p53, DNA repair takes place. If PCNA is rendered non-functional or is absent or present in low quantities in the cell, apoptosis occurs. The evolution from prokaryotes to eukaryotes involved a change of function of PCNA from a 'simple' sliding clamp protein of the DNA polymerase complex to an executive molecule controlling critical cellular decision pathways. The evolution of multicellular organisms led to the development of multicellular processes such as differentiation, senescence and apoptosis. PCNA, already an essential molecule in the life of single cellular organisms, then became a protein critical for the survival of multicellular organisms.