From Processivity to Genome Maintenance: The Many Roles of Sliding Clamps.

Sliding clamps play a pivotal role in the process of replication by increasing the processivity of the replicative polymerase. They also serve as an interacting platform for a plethora of other proteins, which have an important role in other DNA metabolic processes, including DNA repair. In other words, clamps have evolved, as has been correctly referred to, into a mobile "tool-belt" on the DNA, and provide a platform for several proteins that are involved in maintaining genome integrity. Because of the central role played by the sliding clamp in various processes, its study becomes essential and relevant in understanding these processes and exploring the protein as an important drug target. In this review, we provide an updated report on the functioning, interactions, and moonlighting roles of the sliding clamps in various organisms and its utilization as a drug target.

PICH and TOP3A cooperate to induce positive DNA supercoiling.

All known eukaryotic topoisomerases are only able to relieve torsional stress in DNA. Nevertheless, it has been proposed that the introduction of positive DNA supercoiling is required for efficient sister-chromatid disjunction by Topoisomerase 2a during mitosis. Here we identify a eukaryotic enzymatic activity that introduces torsional stress into DNA. We show that the human Plk1-interacting checkpoint helicase (PICH) and Topoisomerase 3a proteins combine to create an extraordinarily high density of positive DNA supercoiling. This activity, which is analogous to that of a reverse-gyrase, is apparently driven by the ability of PICH to progressively extrude hypernegatively supercoiled DNA loops that are relaxed by Topoisomerase 3a. We propose that this positive supercoiling provides an optimal substrate for the rapid disjunction of sister centromeres by Topoisomerase 2a at the onset of anaphase in eukaryotic cells.

Identification and Characterization of an Inside-Out Folding Intermediate of T4 Phage Sliding Clamp.

Protein folding process involves formation of transiently occurring intermediates that are difficult to isolate and characterize. It is both necessary and interesting to characterize the structural conformations adopted by these intermediates, also called molten globules (MG), to understand protein folding. Here, we investigated the equilibrium (un)folding intermediate state of T4 phage gene product 45 (gp45, also known as DNA polymerase processivity factor or sliding clamp) obtained during chemical denaturation. We show that gp45 undergoes substantial conformational rearrangement during unfolding and forms an expanded dry-MG. By monitoring the fluorescence of tryptophans that were strategically introduced at various sites, we demonstrate that the urea-treated molecule has its surface residues flip inside the core, and closely placed residues move farther. We were also able to isolate and purify the MG form of gp45 in native condition (i.e., nondenaturing buffer, at physiological pH and temperature); characteristics of this purified molecule substantially match with urea-treated wild-type gp45. To the best of our knowledge, this is one of the few reports that demonstrate the isolation and purification of a protein folding intermediate in native condition. We believe that our work not only allows us to dissect the process of protein folding, but will also help in the designing of folding inhibitors against sliding clamps to treat a wide variety of diseases from bacterial infection to cancer, due to the vast presence of clamps in all the domains of life.

On the domains of T4 phage sliding clamp gp45: An intermolecular crosstalk governs structural stability and biological activity.

BACKGROUND: DNA polymerase processivity factors are ubiquitously present in all living organisms. Notwithstanding their high significance, the molecular details of clamps pertaining to the factors contributing to their stability are presently lacking. The bacteriophage T4 sliding clamp gp45 forms a homotrimer that besides being involved in DNA replication, moonlights as a transcription factor. Here we have carried out a detailed characterization of gp45 to understand the role of monomer-monomer interface interactions in stability and functioning of the protein. METHODS: We generated several gp45 mutants harboring either Ala or Pro substitutions at the interface residues and performed a detailed investigation using biochemical and biophysical methods including circular dichroism, fluorescence anisotropy and quenching, differential scanning calorimetry, blue-native PAGE, cross-linking, size exclusion chromatography, and dynamic light scattering. We also carried out both transcription and DNA replication to understand the properties of the wild-type and the mutant proteins. RESULTS: One specific mutation S88P leads not only to monomerization, but also results in an unstable molecule. Most interestingly, mutating either Q125 or K164 in the gp45 C-terminal domain negatively affects the stability of the N-terminal domain. We also report that these residues upon mutation to alanine make gp45 inactive for late promoter transcription, whereas strand-displacement DNA replication ability remains unaltered. CONCLUSIONS AND GENERAL SIGNIFICANCE: The results suggest that the two domains of gp45 demonstrate an "inter-monomer" crosstalk that stabilizes the trimer. We also conclude that the residue-specific interactions at the interface allow the protein to function distinctly as replication and transcription factors.

Rapid and Robust PCR-Based All-Recombinant Cloning Methodology.

We report here a PCR-based cloning methodology that requires no post-PCR modifications such as restriction digestion and phosphorylation of the amplified DNA. The advantage of the present method is that it yields only recombinant clones thus eliminating the need for screening. Two DNA amplification reactions by PCR are performed wherein the first reaction amplifies the gene of interest from a source template, and the second reaction fuses it with the designed expression vector fragments. These vector fragments carry the essential elements that are required for the fusion product selection. The entire process can be completed in less than 8 hours. Furthermore, ligation of the amplified DNA by a DNA ligase is not required before transformation, although the procedure yields more number of colonies upon transformation if ligation is carried out. As a proof-of-concept, we show the cloning and expression of GFP, adh, and rho genes. Using GFP production as an example, we further demonstrate that the E. coli T7 express strain can directly be used in our methodology for the protein expression immediately after PCR. The expressed protein is without or with 6xHistidine tag at either terminus, depending upon the chosen vector fragments. We believe that our method will find tremendous use in molecular and structural biology.

Molecular Dissection of the Homotrimeric Sliding Clamp of T4 Phage: Two Domains of a Subunit Display Asymmetric Characteristics.

Sliding clamp proteins are circular dimers or trimers that encircle DNA and serve as processivity factors during DNA replication. Their presence in all the three domains of life and in bacteriophages clearly indicates their high level of significance. T4 gp45, besides functioning as the DNA polymerase processivity factor, also moonlights as the late promoter transcription determinant. Here we report a detailed biophysical analysis of gp45. The chemical denaturation of gp45 probed by circular dichroism spectroscopy, tryptophan fluorescence anisotropy, and blue-native polyacrylamide gel electrophoresis suggests that the protein follows a three-state denaturation profile and displays an intermediate molten globule-like state. The three-state transition was found to be the result of the sequential unfolding of the two domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), of gp45. The experiments involving Trp fluorescence quenching by acrylamide demonstrate that the CTD undergoes substantial changes in conformation during formation of the intermediate state. Further biophysical dissection of the individual domain reveals contrasting properties of the two domains. The NTD unfolds at low urea concentrations and is also susceptible to protease cleavage, whereas the CTD resists urea-mediated denaturation and is not amenable to protease digestion even at higher urea concentrations. These experiments allow us to conclude that the two domains of gp45 differ in their dynamics. While the CTD shows stability and rigidity, we find that the NTD is unstable and flexible. We believe that the asymmetric characteristics of the two domains and the interface they form hold significance in gp45 structure and function.

HDAC5, a key component in temporal regulation of p53-mediated transactivation in response to genotoxic stress.

Despite being one of the most well-studied transcription factors, the temporal regulation of p53-mediated transcription is not very well understood. Recent data suggest that target specificity of p53-mediated transactivation is achieved by posttranslational modifications of p53. K120 acetylation is a modification critical for recruitment of p53 to proapoptotic targets. Our data reveal that histone deacetylase 5 (HDAC5) binds to p53 and abrogates K120 acetylation, resulting in preferential recruitment of p53 to proarrest and antioxidant targets at early phases of stress. However, upon prolonged genotoxic stress, HDAC5 undergoes nuclear export. Concomitantly, p53 is acetylated at the K120 residue and selectively transactivates proapoptotic target genes, leading to onset of apoptosis. Furthermore, upon genotoxic stress in mice where HDAC5 expression is downregulated, the onset of apoptosis is accelerated in the highly vulnerable tissues. These findings suggest that HDAC5 is a key determinant of p53-mediated cell fate decisions in response to genotoxic stress.

Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options.

We report the designing of three expression vectors that can be used for rapid cloning of any blunt-end DNA segment. Only a single set of oligonucleotides are required to perform the amplification of the target DNA and its cloning in all three vectors simultaneously. The DNA thus cloned can express a protein either with or without a hexa-histidine tag depending upon the vector used. The expression occurs from T7 promoter when transformed into E. coli BL21(DE3). Two of the three plasmids have been designed to provide the expressed protein with either N- or C-terminus 6 histidine amino acids in tandem. The third plasmid, however, does not add any tag to the expressed protein. The cloning is achieved quickly with the requirement of phosphorylation of PCR product without any restriction digestion. Additionally, the generated clones can be confirmed with a single step PCR reaction carried out from bacterial colonies (generally termed as "colony PCR"). We show the cloning, expression and purification of Green Fluorescent Protein (GFP) as proof-of-concept. Additionally, we also show the cloning and expression of four sigma factors from Mycobacterium tuberculosis further demonstrating the utility of the designed plasmids. We strongly believe that the vectors and the strategy that we have developed will facilitate the rapid cloning and expression of any gene in E. coli BL21(DE3) with or without a hexa-histidine tag.