Biomedical subjects

Jeff Finkelstein

Publications and source records attributed to Jeff Finkelstein.

4 recordsLinked to original sources

PubMed2004

Dual functions, clamp opening and primer-template recognition, define a key clamp loader subunit.

Clamp loader proteins catalyze assembly of circular sliding clamps on DNA to enable processive DNA replication. During the reaction, the clamp loader binds primer-template DNA and positions it in the center of a clamp to form a topological link between the two. Clamp loaders are multi-protein complexes, such as the five protein Escherichia coli, Saccharomyces cerevisiae, and human clamp loaders, and the two protein Pyrococcus furiosus and Methanobacterium thermoautotrophicum clamp loaders, and thus far the site(s) responsible for binding and selecting primer-template DNA as the target for clamp assembly remain unknown. To address this issue, we analyzed the interaction between the E.coli gamma complex clamp loader and DNA using UV-induced protein-DNA cross-linking and mass spectrometry. The results show that the delta subunit in the gamma complex makes close contact with the primer-template junction. Tryptophan 279 in the delta C-terminal domain lies near the 3'-OH primer end and may play a key role in primer-template recognition. Previous studies have shown that delta also binds and opens the beta clamp (hydrophobic residues in the N-terminal domain of delta contact beta. The clamp-binding and DNA-binding sites on delta appear positioned for facile entry of primer-template into the center of the clamp and exit of the template strand from the complex. A similar analysis of the S.cerevisiae RFC complex suggests that the dual functionality observed for delta in the gamma complex may be true also for clamp loaders from other organisms.

Adenosine Triphosphate↗

PubMed2004

Crystal structure of the chi:psi sub-assembly of the Escherichia coli DNA polymerase clamp-loader complex.

The chi (chi) and psi (psi) subunits of Escherichia coli DNA polymerase III form a heterodimer that is associated with the ATP-dependent clamp-loader machinery. In E. coli, the chi:psi heterodimer serves as a bridge between the clamp-loader complex and the single-stranded DNA-binding protein. We determined the crystal structure of the chi:psi heterodimer at 2.1 A resolution. Although neither chi (147 residues) nor psi (137 residues) bind to nucleotides, the fold of each protein is similar to the folds of mononucleotide-(chi) or dinucleotide-(psi) binding proteins, without marked similarity to the structures of the clamp-loader subunits. Genes encoding chi and psi proteins are found to be readily identifiable in several bacterial genomes and sequence alignments showed that residues at the chi:psi interface are highly conserved in both proteins, suggesting that the heterodimeric interaction is of functional significance. The conservation of surface-exposed residues is restricted to the interfacial region and to just two other regions in the chi:psi complex. One of the conserved regions was found to be located on chi, distal to the psi interaction region, and we identified this as the binding site for a C-terminal segment of the single-stranded DNA-binding protein. The other region of sequence conservation is localized to an N-terminal segment of psi (26 residues) that is disordered in the crystal structure. We speculate that psi is linked to the clamp-loader complex by this flexible, but conserved, N-terminal segment, and that the chi:psi unit is linked to the single-stranded DNA-binding protein via the distal surface of chi. The base of the clamp-loader complex has an open C-shaped structure, and the shape of the chi:psi complex is suggestive of a loose docking within the crevice formed by the open faces of the delta and delta' subunits of the clamp-loader.

Adenosine Triphosphate↗

PubMed2003

Replication factor C clamp loader subunit arrangement within the circular pentamer and its attachment points to proliferating cell nuclear antigen.

Replication factor C (RFC) is a heteropentameric AAA+ protein clamp loader of the proliferating cell nuclear antigen (PCNA) processivity factor. The prokaryotic homologue, gamma complex, is also a heteropentamer, and structural studies show the subunits are arranged in a circle. In this report, Saccharomyces cerevisiae RFC protomers are examined for their interaction with each other and PCNA. The data lead to a model of subunit order around the circle. A characteristic of AAA+ oligomers is the use of bipartite ATP sites in which one subunit supplies a catalytic arginine residue for hydrolysis of ATP bound to the neighboring subunit. We find that the RFC(3/4) complex is a DNA-dependent ATPase, and we use this activity to determine that RFC3 supplies a catalytic arginine to the ATP site of RFC4. This information, combined with the subunit arrangement, defines the composition of the remaining ATP sites. Furthermore, the RFC(2/3) and RFC(3/4) subassemblies bind stably to PCNA, yet neither RFC2 nor RFC4 bind tightly to PCNA, indicating that RFC3 forms a strong contact point to PCNA. The RFC1 subunit also binds PCNA tightly, and we identify two hydrophobic residues in RFC1 that are important for this interaction. Therefore, at least two subunits in RFC make strong contacts with PCNA, unlike the Escherichia coli gamma complex in which only one subunit makes strong contact with the beta clamp. Multiple strong contact points to PCNA may reflect the extra demands of loading the PCNA trimeric ring onto DNA compared with the dimeric beta ring.

Adenosine Triphosphatases↗

PubMed2003

Overproduction and analysis of eukaryotic multiprotein complexes in Escherichia coli using a dual-vector strategy.

Biochemical studies of eukaryotic proteins are often constrained by low availability of these typically large, multicomponent protein complexes in pure form. Escherichia coli is a commonly used host for large-scale protein production; however, its utility for eukaryotic protein production is limited because of problems associated with transcription, translation, and proper folding of proteins. Here we describe the development and testing of pLANT, a vector that addresses many of these problems simultaneously. The pLANT vector contains a T7 promoter-controlled expression unit, a p15A origin of replication, and genes for rare transfer RNAs and kanamycin resistance. Thus, the pLANT vector can be used in combination with the pET vector to coexpress multiple proteins in E. coli. Using this approach, we have successfully produced high-milligram quantities of two different Saccharomyces cerevisiae complexes in E. coli: the heterodimeric Msh2-Msh6 mismatch repair protein (248kDa) and the five-subunit replication factor C clamp loader (250 kDa). Quantitative analyses indicate that these proteins are fully active, affirming the utility of pLANT+pET-based production of eukaryotic proteins in E. coli for in vitro studies of their structure and function.

DNA-Binding Proteins↗