Significance DNA damage can be a potent block to replication. One pathway bypass is translesion synthesis (TLS) by specialized polymerases. These conserved TLS polymerases have higher error rates than replicative polymerases, requiring careful regulation of polymerase exchange. By reconstituting the full exchange reaction at single-molecule level, we show how distinct sets binding sites on β processivity clamp regulate between Escherichia coli (Pol) III and Pol IV. At low concentrations, IV...

Abstract Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding roles regulations site‐specific glycosylation events remains a significant challenge due several technological limitations. These limitations include lack available tools biochemical characterization enzymes involved in glycosylation. A particular synthesis oligosaccharyltransferases (OSTs), which catalyze glycans...

Affinity capture (AC) combined with mass spectrometry (MS)-based proteomics is highly utilized throughout the drug discovery pipeline to determine small-molecule target selectivity and engagement. However, tedious sample preparation steps time-consuming MS acquisition process have limited its use in a high-throughput format. Here, we report an automated workflow employing biotinylated probes streptavidin magnetic beads for enrichment 96-well plate format, ending direct sampling from EvoSep...

Abstract Unrepaired DNA lesions are a potent block to replication, leading replication fork collapse, double-strand breaks, and cell death. Error-prone polymerases overcome this blockade by synthesizing past in process called translesion synthesis (TLS), but how TLS gain access the template remains poorly understood. In study, we use particle-tracking PALM image live Escherichia coli cells containing functional fusion of endogenous copy Pol IV photoactivatable fluorescent protein PAmCherry....

Escherichia coli has three DNA polymerases implicated in the bypass of damage, a process called translesion synthesis (TLS) that alleviates replication stalling. Although these are specialized for different lesions, it is unclear if they interact differently with machinery. Of three, polymerase (Pol) II remains most enigmatic. Here we report stable ternary complex Pol II, replicative III core and dimeric processivity clamp, β. Single-molecule experiments reveal interactions β allow rapid...

Translesion DNA synthesis (TLS) by specialized polymerases (Pols) is a conserved mechanism for tolerating replication blocking lesions. The actions of TLS Pols are managed in part ring-shaped sliding clamp proteins. In addition to catalyzing TLS, altered expression impedes cellular growth. goal this study was define the relationship between physiological function Escherichia coli Pol IV and its ability impede growth when overproduced. To end, 13 novel mutants were identified that failed...

Enzyme rates are usually considered to be dependent on local properties of the molecules involved in reactions. However, for large molecules, distant constraints might affect reaction by affecting dynamics leading transition states. In single-molecule experiments we have found that enzymes relax DNA torsional stress display depend strongly how ends molecule constrained; with different-sized particles tethered end 10-kb DNAs reveal enzyme inversely correlated particle drag coefficients. This...

Significance DNA replication is the high-fidelity process by which cells duplicate their chromosomes prior to cell division. Cellular constantly damaged, and resulting lesions can block replication, leading genome instability death. Cells use multiple pathways resolve stalled replication. Understanding resolution pathway choice important because some of these are more likely introduce mutations than others. In bacterial cells, stresses, including antibiotic treatment, lead mutagenesis, may...

Structural proteins such as "suckerins" present promising avenues for fabricating functional materials. Suckerins are a family of naturally occurring block copolymer-type that comprise the sucker ring teeth cephalopods and known to self-assemble into supramolecular networks nanoconfined β-sheets. Here, we report characterization controllable, nanoscale self-assembly suckerin-12 (S12). We characterize impacts salt, pH, protein concentration on S12 solubility, secondary structure,...

Abstract Affinity capture (AC) combined with mass spectrometry (MS)-based proteomics is highly utilized throughout the drug discovery pipeline to determine small molecule target selectivity and engagement. However, tedious sample preparation steps time-consuming MS acquisition process has limited its use in high-throughput format. Here, we report an automated workflow employing biotinylated probes streptavidin magnetic beads for enrichment 96-well plate format, ending direct sampling from...

Abstract Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding roles regulations site-specific glycosylation events remains a significant challenge due several technological limitations. These limitations include lack available tools biochemical characterization enzymes involved in glycosylation. A particular synthesis oligosaccharyltransferases (OSTs), which catalyze glycans...

The field of chemical biology has introduced several approaches, typically using probes, to measure the direct binding interaction a small molecule with its biological target in cells. use these engagement assays pharmaceutical development can support mechanism action hypothesis testing, rank ordering compounds, and iterative improvements matter. This Feature Article highlights newer application approaches: quantification animal models late stage preclinical nomination drug candidate...

Abstract Structural proteins such as the “suckerins” present promising avenues for fabricating functional materials. Suckerins are a family of naturally occurring block copolymer-type that comprise sucker ring teeth cephalopods and known to self-assemble into supramolecular networks nanoconfined β -sheets. Here, we report characterization controllable, nanoscale self-assembly suckerin-12 (S12). We characterize impacts salt, pH, protein concentration on S12 solubility, secondary structure,...

Translesion synthesis (TLS) alleviates replication stalling at DNA lesions. Ring‐shaped processivity clamps play a critical but ill‐defined role in mediating exchange between replicative and TLS polymerases sites of damage. We have developed single‐molecule approach to reconstitute the Escherichia coli polymerase, Pol III, with II IV. observe clamp‐mediated III both polymerases, that distinct sets interactions each polymerase clamp govern mode exchange. These results suggest selection is not...

Abstract The cellular thermal shift assay (CETSA®) is a target engagement method widely used for preclinical characterization of small molecule compounds. CETSA® has been semi-quantitative readouts in whole blood with PBMC isolation, and quantitative, plate-based using cell lines. However, there no quantitative evaluation unprocessed human blood, which preferred clinical applications. Here we report two separate formats – Alpha MSD that require less than 100 μL per sample without isolation....

The cellular thermal shift assay (CETSA®) is a target engagement method widely used for preclinical characterization of small molecule compounds. CETSA® has been semi-quantitative readouts in whole blood with PBMC isolation, and quantitative, plate-based using cell lines. However, there no quantitative evaluation unprocessed human blood, which preferred clinical applications. Here we report two separate formats – Alpha MSD that require less than 100 μL per sample without isolation. We chose...

Abstract DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes reprime synthesis downstream lesion, creating single-stranded gap that repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within bacterial determine pathway lesion-stalled replisomes. This selection controlled...