Precision screening facilitates clinical classification of BRCA2-PALB2 binding variants with benign and pathogenic functional effects.

BACKGROUNDDecoding the clinical impact of genetic variants is particularly important for precision medicine in cancer. Genetic screening of mainly patients with breast and ovarian cancer has identified numerous BRCA1/BRCA2 variants of uncertain significance (VUS) that remain unclassified owing to a lack of pedigrees and functional data.METHODSHere, we used CRISPR-Select - a technology that exploits unique inbuilt controls at the endogenous locus - to assess 54 rare ClinVar VUS located in the PALB2-binding domain of BRCA2.

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.

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.

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.

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.

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.

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.

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.

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.