Biomedical subjects

Roxana E Georgescu

Publications and source records attributed to Roxana E Georgescu.

8 recordsLinked to original sources

PubMed2006

Crystal structure of the catalytic alpha subunit of E. coli replicative DNA polymerase III.

Bacterial replicative DNA polymerases such as Polymerase III (Pol III) share no sequence similarity with other polymerases. The crystal structure, determined at 2.3 A resolution, of a large fragment of Pol III (residues 1-917), reveals a unique chain fold with localized similarity in the catalytic domain to DNA polymerase beta and related nucleotidyltransferases. The structure of Pol III is strikingly different from those of members of the canonical DNA polymerase families, which include eukaryotic replicative polymerases, suggesting that the DNA replication machinery in bacteria arose independently. A structural element near the active site in Pol III that is not present in nucleotidyltransferases but which resembles an element at the active sites of some canonical DNA polymerases suggests that, at a more distant level, all DNA polymerases may share a common ancestor. The structure also suggests a model for interaction of Pol III with the sliding clamp and DNA.

Bacterial Proteins↗

PubMed2005

Conserved interactions in the Staphylococcus aureus DNA PolC chromosome replication machine.

The PolC holoenzyme replicase of the Gram-positive Staphylococcus aureus pathogen has been reconstituted from pure subunits. We compared individual S. aureus replicase subunits with subunits from the Gram-negative Escherichia coli polymerase III holoenzyme for activity and interchangeability. The central organizing subunit, tau, is smaller than its Gram-negative homolog, yet retains the ability to bind single-stranded DNA and contains DNA-stimulated ATPase activity comparable with E. coli tau. S. aureus tau also stimulates PolC, although they do not form as stabile a complex as E. coli polymerase III.tau. We demonstrate that the extreme C-terminal residues of PolC bind to and function with beta clamps from different bacteria. Hence, this polymerase-clamp interaction is highly conserved. Additionally, the S. aureus delta wrench of the clamp loader binds to E. coli beta. The S. aureus clamp loader is even capable of loading E. coli and Streptococcus pyogenes beta clamps onto DNA. Interestingly, S. aureus PolC lacks functionality with heterologous beta clamps when they are loaded onto DNA by the S. aureus clamp loader, suggesting that the S. aureus clamp loader may have difficulty ejecting from heterologous clamps. Nevertheless, these overall findings underscore the conservation in structure and function of Gram-positive and Gram-negative replicases despite >1 billion years of evolutionary distance between them.

Bacterial Proteins↗

PubMed2004

Protein trafficking on sliding clamps.

The sliding clamps of chromosomal replicases are acted upon by both the clamp loader and DNA polymerase. Several other proteins and polymerases also interact with the clamp. These proteins bind the clamp at the same spot and use it in sequential fashion. First the clamp loader must bind the clamp in order to load it onto DNA, but directly thereafter the clamp loader must clear away from the clamp so it can be used by the replicative DNA polymerase. At the end of replication, the replicase is ejected from the clamp, which presumably allows the clamp to interact with yet other proteins after its use by the replicase. This paper describes how different proteins in the Escherichia coli replicase, DNA polymerase III holoenzyme, coordinate their traffic flow on the clamp. The mechanism by which traffic flow on the beta clamp is directed is based on competition of the proteins for the clamp, where DNA structure modulates the competition. It seems likely that the principles will generalize to a traffic flow of other factors on these circular clamp proteins.

Binding, Competitive↗

PubMed2003

Competitive processivity-clamp usage by DNA polymerases during DNA replication and repair.

Protein clamps are ubiquitous and essential components of DNA metabolic machineries, where they serve as mobile platforms that interact with a large variety of proteins. In this report we identify residues that are required for binding of the beta-clamp to DNA polymerase III of Escherichia coli, a polymerase of the Pol C family. We show that the alpha polymerase subunit of DNA polymerase III interacts with the beta-clamp via its extreme seven C-terminal residues, some of which are conserved. Moreover, interaction of Pol III with the clamp takes place at the same site as that of the delta-subunit of the clamp loader, providing the basis for a switch between the clamp loading machinery and the polymerase itself. Escherichia coli DNA polymerases I, II, IV and V (UmuC) interact with beta at the same site. Given the limited amounts of clamps in the cell, these results suggest that clamp binding may be competitive and regulated, and that the different polymerases may use the same clamp sequentially during replication and repair.

Amino Acid Sequence↗

PubMed2003

A peptide switch regulates DNA polymerase processivity.

Chromosomal DNA polymerases are tethered to DNA by a circular sliding clamp for high processivity. However, lagging strand synthesis requires the polymerase to rapidly dissociate on finishing each Okazaki fragment. The Escherichia coli replicase contains a subunit (tau) that promotes separation of polymerase from its clamp on finishing DNA segments. This report reveals the mechanism of this process. We find that tau binds the C-terminal residues of the DNA polymerase. Surprisingly, this same C-terminal "tail" of the polymerase interacts with the beta clamp, and tau competes with beta for this sequence. Moreover, tau acts as a DNA sensor. On binding primed DNA, tau releases the polymerase tail, allowing polymerase to bind beta for processive synthesis. But on sensing the DNA is complete (duplex), tau sequesters the polymerase tail from beta, disengaging polymerase from DNA. Therefore, DNA sensing by tau switches the polymerase peptide tail on and off the clamp and coordinates the dynamic turnover of polymerase during lagging strand synthesis.

Animals↗

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↗

PubMed2002

The DnaC helicase loader is a dual ATP/ADP switch protein.

Helicases are transferred to replication origins by helicase loading factors. The Escherichia coli DnaC and eukaryotic Cdc6/18 helicase loaders contain ATP sites and are both members of the AAA+ family. One might expect that ATP is required for helicase loading; however, this study on DnaC illustrates that ATP is not actually needed for DnaC to load helicase onto single-strand DNA (ssDNA). In fact, it seems to be a paradox that after transfer of helicase to DNA, DnaC-ATP inhibits helicase action. In addition, ATP is required for DnaC function at an early step in oriC replication in which ATP stimulates ssDNA binding by DnaC, leading to expansion of the ssDNA bubble at the origin. Two cofactors, ssDNA and DnaB, trigger hydrolysis of ATP, converting DnaC to the ADP form that no longer inhibits DnaB. These observations have led to the idea that DnaC is a 'dual' switch protein, where both the ATP and the ADP forms are sequentially required for replication. This dual switching process may underlie the sensitivity of DnaB to even small fluctuations in DnaC levels.

Adenosine Diphosphate↗

PubMed2002

Combining conformational flexibility and continuum electrostatics for calculating pK(a)s in proteins.

Protein stability and function relies on residues being in their appropriate ionization states at physiological pH. In situ residue pK(a)s also provides a sensitive measure of the local protein environment. Multiconformation continuum electrostatics (MCCE) combines continuum electrostatics and molecular mechanics force fields in Monte Carlo sampling to simultaneously calculate side chain ionization and conformation. The response of protein to charges is incorporated both in the protein dielectric constant (epsilon(prot)) of four and by explicit conformational changes. The pK(a) of 166 residues in 12 proteins was determined. The root mean square error is 0.83 pH units, and >90% have errors of <1 pH units whereas only 3% have errors >2 pH units. Similar results are found with crystal and solution structures, showing that the method's explicit conformational sampling reduces sensitivity to the initial structure. The outcome also changes little with protein dielectric constant (epsilon(prot) 4-20). Multiconformation continuum electrostatics titrations show coupling of conformational flexibility and changes in ionization state. Examples are provided where ionizable side chain position (protein G), Asn orientation (lysozyme), His tautomer distribution (RNase A), and phosphate ion binding (RNase A and H) change with pH. Disallowing these motions changes the calculated pK(a).

Animals↗