Flap endonuclease-1 (FEN1) is a key enzyme for maintaining genomic stability and replication. Proliferating cell nuclear antigen (PCNA) binds FEN1 and stimulates its endonuclease activity. The structural basis of the FEN1-PCNA interaction was revealed by the crystal structure of the complex between human FEN1 and PCNA. The main interface involves the C-terminal tail of FEN1, which forms two b-strands connected by a short helix, the bA-aA-bB motif, participating in b-b and hydrophobic interactions with PCNA. These interactions are similar to those previously observed for the p21 CIP1/WAF1 peptide. However, this structure involving the full-length enzyme has revealed additional interfaces that are involved in the core domain. The interactions at the interfaces maintain the enzyme in an inactive 'locked-down' orientation and might be utilized in rapid DNA-tracking by preserving the central hole of PCNA for sliding along the DNA. A hinge region present between the core domain and the C-terminal tail of FEN1 would play a role in switching the FEN1 orientation from an inactive to an active orientation.

The consequence of unpaired electrons in organic molecules has fascinated and confounded chemists for over a century. The study of open-shell molecules has been rekindled in recent years as new synthetic methods, improved spectroscopic techniques and powerful computational tools have been brought to bear on this field. Nonetheless, it is the intrinsic instability of the biradical species that limits the practicality of this research. Here we report the synthesis and characterization of a molecule based on the diindeno[b,i]anthracene framework that exhibits pronounced open-shell character yet possesses remarkable stability. The synthetic route is rapid, efficient and possible on the gram scale. The molecular structure was confirmed through single-crystal X-ray diffraction. From variable-temperature Raman spectroscopy and magnetic susceptibility measurements a thermally accessible triplet excited state was found. Organic field-effect transistor device data show an ambipolar performance with balanced electron and hole mobilities. Our results demonstrate the rational design and synthesis of an air- and temperature-stable biradical compound.

A major nidogen binding site of mouse laminin was previously localized to about three EGF‐like repeats (Nos 3–5) of its B2 chain domain III [M. Gerl et al. (1991) Eur. J. Biochem., 202, 167]. The corresponding cDNA was amplified by polymerase chain reaction and inserted into a eukaryotic expression vector tagged with a signal peptide. Stably transfected human kidney cell clones were shown to process and secrete the resulting fragment B2III3‐5 in substantial quantities. It possessed high binding activity for recombinant nidogen in ligand assays, with an affinity comparable with that of authentic laminin fragments. In addition, complexes of B2III3‐5 and nidogen could be efficiently converted into a covalent complex by cross‐linking reagents. Proteolytic degradation of the covalent complex demonstrated the association of B2III3‐5 with a approximately 80 residue segment of nidogen domain G3 to which laminin binding has previously been attributed. The correct formation of most of the 12 disulfide bridges in B2III3‐5 was indicated from its protease resistance and the complete loss of cross‐reacting epitopes as well as of nidogen‐binding activity after reduction and alkylation. Smaller fragments were prepared by the same recombinant procedure and showed that combinations of EGF‐like repeats 3–4 and 4–5 and the single repeat 4 but not repeats 3 or 5 possess full nidogen‐binding activity. This identifies repeat 4 as the only binding structure. The sequence of repeat 4 is well conserved in the human and in part in the Drosophila laminin B2 chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferating cell nuclear antigen (PCNA) is essential for eukaryotic DNA replication and functions as a processivity factor of DNA polymerase ␦ (pol ␦). Due to the functional and structural similarity with the ␤-subunit of Escherichia coli DNA polymerase III, it has been proposed that PCNA would act as a molecular clamp during DNA synthesis. By site-directed mutagenesis and biochemical analyses, we have studied the functional domains of human PCNA required for stimulation of replication factor C (RF-C) ATPase and DNA synthesis by pol ␦. Short deletions from either the N or C termini caused drastic changes in extraction and chromatographic behaviors, suggesting that both of these terminal regions are crucial to fold the tertiary structure of PCNA. The short C-terminal stretch from Lys 254 to Glu 256 is necessary for stimulation of RF-C ATPase activity, but not for stimulation of DNA synthesis by pol ␦. Nine basic amino acids that are essential for activating DNA synthesis by pol ␦ are positioned at the internal ␣-helices of PCNA. This result is in good agreement with the observation that PCNA has a ring structure similar to the ␤-subunit and clamps a template DNA through this positively charged internal surface. Several other charged amino acids are also required to stimulate either RF-C ATPase or pol ␦ DNA synthesis. Some of them are positioned at loops which are exposed on one of the side surface of PCNA adjacent to the C-terminal loop. In addition, the ␤-sheets composing the intermolecular interface of the trimeric PCNA are important for interaction with pol ␦. Therefore, the outer surface of PCNA has multiple functional surfaces which are responsible for the interaction with multiple factors. Furthermore, the two side surfaces seem to be functionally distinguishable, and this may determine the orientation of tracking PCNA along the DNA.The proliferating cell nuclear antigen (PCNA) 1 is an essential replication factor for simian virus 40 (SV40) DNA replication in vitro and is involved in the elongation stages of DNA replication (1-4). Recent studies have further demonstrated the involvement of PCNA in cellular chromosomal DNA replication in vivo (5-7). In addition to its essential role in DNA replication, PCNA is required for nucleotide excision repair of DNA (8 -10) and also may participate in the cell cycle control as demonstrated by an interaction with a cyclin dependent kinase complex (11). Therefore, PCNA is multifunctional through the interaction with several specific partners, and all of the functions are crucial for cell proliferation.Genes encoding PCNA have been isolated from various eukaryotes and are composed of highly conserved amino acid sequences of around 260 residues. The functions of PCNA during DNA replication have been elucidated by studies of SV40 DNA replication in vitro. In this reaction, three protein components, PCNA, DNA polymerase (pol) ␦, and replication factor C (RF-C) are required for leading strand DNA synthesis following the initiation of DNA synthesis by DNA pol ␣ at the SV4...