DNA transactions such as DNA replication and DNA repair require the concerted action of many enzymes, together with other proteins and non-protein cofactors. Among them three main accessory proteins, replication factor C (RF-C), proliferating-cell nuclear antigen (PCNA) and replication protein A (RP-A), are essential for accurate and processive DNA synthesis by DNA polymerases. RF-C is a complex consisting of five polypeptides with distinct functions. RF-C can bind to a template-primer junction and, in the presence of ATP, load the PCNA clamp onto DNA, thereby recruiting DNA polymerases to the site of DNA synthesis. RF-C not only acts as a clamp loader in DNA replication and DNA repair, but there is some evidence that it could be involved in several other processes such as transcription, S-phase checkpoint regulation, apoptosis, differentiation and telomere-length regulation.Keywords : eukaryotic DNA replication; replication factor C; proliferating-cell nuclear antigen; clamp; clamp loader ; DNA polymerase δ ; S-phase checkpoint regulation ; apoptosis; differentiation; telomere. Of clamps and clamp loadersrespective clamps onto DNA. High sequence similarity between the clamp loaders suggest that their mechanisms of action may DNA replication, one of the most remarkable and challeng-be very similar [4]. The eukaryotic RF-C clamp loader consists ing cellular events, is the result of the collaboration of a formida-of five distinct subunits (Table 1), as does the E. coli γ complex. ble amount of proteins. The mechanisms at the basis of this By loading the homotrimer proliferating-cell nuclear antigen complicated event are similar in prokaryotes and eukaryotes, de-(PCNA) clamp onto DNA, it confers high processivity to polyspite the evolutionary distance. In each of these cell types, the merases δ and ε. Despite there being virtually no similarity in replicative polymerases are chaperoned by several accessory sequence between β and PCNA, the three-dimensional structures proteins that confer speed and high processivity.are almost identical [5]. The amino acid sequence of PCNA is Responsible for DNA replication in Escherichia coli is the only approximately 70% the length of that of β, so that three DNA polymerase III holoenzyme, a ten-subunit complex (Ta-monomers of PCNA are required to form the typical doughnutble 1) [1]. The DNA polymerase (polymerase III core) consists like structure, in contrast to β where only two monomers are of A (the polymerase), ε (the proofreading exonuclease) and θ enough. The T4 gene 44/62 protein complex contains five subsubunits. The clamp loader, called γ complex, contains five subunits, four protomers of the gene 44 protein and one copy of the units, γ, δ, δ′, ψ and χ, and is required for the ATP-dependent gene 62 protein [6]. It is required for the loading of the product assembly of the β clamp onto DNA. Contrary to all eukaryotes of gene 45, the T4 bacteriophage counterpart of PCNA and E. and the T4 phage, E. coli contains an additional connector procoli β. Despite the apparent similar...

Replication factor C (RF‐C), a complex of five polypeptides, is essential for cell‐free SV40 origin‐dependent DNA replication and viability in yeast. The cDNA encoding the large subunit of human RF‐C (RF‐Cp145) was cloned in a Southwestern screen. Using deletion mutants of RF‐Cp145 we have mapped the DNA binding domain of RF‐Cp145 to amino acid residues 369–480. This domain is conserved among both prokaryotic DNA ligases and eukaryotic poly(ADP‐ribose) polymerases and is absent in other subunits of RF‐C. The PCNA binding domain maps to amino acid residues 481–728 and is conserved in all five subunits of RF‐C. The PCNA binding domain of RF‐Cp145 inhibits several functions of RF‐C, such as: (i) in vitro DNA replication of SV40 origin‐containing DNA; (ii) RF‐C‐dependent loading of PCNA onto DNA; and (iii) RF‐C‐dependent DNA elongation. The PCNA binding domain of RF‐Cp145 localizes to the nucleus and inhibits DNA synthesis in transfected mammalian cells. In contrast, the DNA binding domain of RF‐Cp145 does not inhibit DNA synthesis in vitro or in vivo. We therefore conclude that amino acid residues 481–728 of human RF‐Cp145 are critical and act as a dominant negative mutant of RF‐C function in DNA replication in vivo.

We have previously described a 160-bp enhancer (BCE-1) in the bovine ␤-casein gene that is activated in the presence of prolactin and extracellular matrix (ECM). Here we report the characterization of the enhancer by deletion and site-directed mutagenesis, electrophoretic mobility shift analysis, and in vivo footprinting. Two essential regions were identified by analysis of mutant constructions: one binds C/EBP-␤ and the other binds MGF/STAT5 and an as-yet-unidentified binding protein. However, no qualitative or quantitative differences in the binding of these proteins were observed in electrophoretic mobility shift analysis using nuclear extracts derived from cells cultured in the presence or absence of ECM with or without prolactin, indicating that prolactin-and ECM-induced transcription was not dependent on the availability of these factors in the functional cell lines employed. An in vivo footprinting analysis of the factors bound to nuclear chromatin in the presence or absence of ECM and/or prolactin found no differences in the binding of C/EBP-␤ but did not provide definitive results for the other factors. Neither ECM nor prolactin activated BCE-1 in transient transfections, suggesting that the chromosomal structure of the integrated template may be required for ECM-induced transcription. Further evidence is that treatment of cells with inhibitors of histone deacetylase was sufficient to induce transcription of integrated BCE-1 in the absence of ECM. Together, these results suggest that the ECM induces a complex interaction between the enhancer-bound transcription factors, the basal transcriptional machinery, and a chromosomally integrated template responsive to the acetylation state of the histones.It is now well established that the processes of development and differentiation depend on a cell's ability to correctly perceive its microenvironment (reviewed in references 1 and 43). A key component of this environment is the extracellular matrix (ECM). The ECM is an organized network of glycoproteins, proteoglycans, and glycosaminoglycans, components important for cell morphology as well as for signal transduction via cell surface integrins and ultimately for tissue-specific gene expression (reviewed in reference 43).The mammary gland appears to be particularly well suited for the study of ECM-induced differentiation and gene expression. In the adult animal, the gland develops after puberty and functionally differentiates in response to pregnancy. The mechanisms involved in these developmental processes are complex and guided by various hormones (54), growth factors (53), and the ECM (3). Milk protein expression is initiated at mid-pregnancy and correlates with the synthesis and deposition of a specialized laminin-rich ECM during alveolar development. Expression of these milk proteins can be used as markers for the differentiated state of the gland. In the last decade, a number of model systems using mammary epithelial cells to study ECM-dependent gene regulation have been developed. These range from primar...

Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and repair. It is a molecular matchmaker required for loading of proliferating cell nuclear antigen (PCNA) onto double-stranded DNA and, thus, for PCNA-dependent DNA elongation by DNA polymerases ␦ and ⑀. To elucidate the mode of RF-C binding to the PCNA clamp, modified forms of human PCNA were used that could be 32 P-labeled in vitro either at the C or the N terminus. Using a kinase protection assay, we show that the heteropentameric calf thymus RF-C was able to protect the C-terminal region but not the N-terminal region of human PCNA from phosphorylation, suggesting that RF-C interacts with the PCNA face at which the C termini are located (C-side). A similar protection profile was obtained with the recently identified PCNA binding region (residues 478 -712), but not with the DNA binding region (residues 366 -477), of the human RF-C large subunit (Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messner, H., Khastilba, S., Hü bscher, U., and Fotedar, A., (1996) EMBO J., 15, 4423-4433). Furthermore, we show that the RF-C 36 kDa subunit of human RF-C could interact independently with the C-side of PCNA. The RF-C large subunit from a third species, namely Drosophila melanogaster, interacted similarly with the modified human PCNA, indicating that the interaction between RF-C and PCNA is conserved through eukaryotic evolution.