A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes.

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 can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within the Escherichia coli replisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of translesion synthesis (TLS) polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes.

A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases.

Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 420 µg/ml for the single-subunit OST enzyme, "Protein glycosylation B" (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.

Exchange between Escherichia coli polymerases II and III on a processivity clamp.

Escherichia coli has three DNA polymerases implicated in the bypass of DNA damage, a process called translesion synthesis (TLS) that alleviates replication stalling. Although these polymerases are specialized for different DNA lesions, it is unclear if they interact differently with the replication machinery. Of the three, DNA polymerase (Pol) II remains the most enigmatic. Here we report a stable ternary complex of Pol II, the replicative polymerase Pol III core complex and the dimeric processivity clamp, ß. Single-molecule experiments reveal that the interactions of Pol II and Pol III with ß allow for rapid exchange during DNA synthesis. As with another TLS polymerase, Pol IV, increasing concentrations of Pol II displace the Pol III core during DNA synthesis in a minimal reconstitution of primer extension. However, in contrast to Pol IV, Pol II is inefficient at disrupting rolling-circle synthesis by the fully reconstituted Pol III replisome. Together, these data suggest a ß-mediated mechanism of exchange between Pol II and Pol III that occurs outside the replication fork.

A Genetic Selection for dinB Mutants Reveals an Interaction between DNA Polymerase IV and the Replicative Polymerase That Is Required for Translesion Synthesis.

Translesion DNA synthesis (TLS) by specialized DNA polymerases (Pols) is a conserved mechanism for tolerating replication blocking DNA lesions. The actions of TLS Pols are managed in part by ring-shaped sliding clamp proteins. In addition to catalyzing TLS, altered expression of TLS Pols impedes cellular growth. The goal of this study was to define the relationship between the physiological function of Escherichia coli Pol IV in TLS and its ability to impede growth when overproduced. To this end, 13 novel Pol IV mutants were identified that failed to impede growth. Subsequent analysis of these mutants suggest that overproduced levels of Pol IV inhibit E. coli growth by gaining inappropriate access to the replication fork via a Pol III-Pol IV switch that is mechanistically similar to that used under physiological conditions to coordinate Pol IV-catalyzed TLS with Pol III-catalyzed replication. Detailed analysis of one mutant, Pol IV-T120P, and two previously described Pol IV mutants impaired for interaction with either the rim (Pol IVR) or the cleft (Pol IVC) of the ß sliding clamp revealed novel insights into the mechanism of the Pol III-Pol IV switch. Specifically, Pol IV-T120P retained complete catalytic activity in vitro but, like Pol IVR and Pol IVC, failed to support Pol IV TLS function in vivo. Notably, the T120P mutation abrogated a biochemical interaction of Pol IV with Pol III that was required for Pol III-Pol IV switching. Taken together, these results support a model in which Pol III-Pol IV switching involves interaction of Pol IV with Pol III, as well as the ß clamp rim and cleft. Moreover, they provide strong support for the view that Pol III-Pol IV switching represents a vitally important mechanism for regulating TLS in vivo by managing access of Pol IV to the DNA.

Polymerase exchange on single DNA molecules reveals processivity clamp control of translesion synthesis.

Translesion synthesis (TLS) by Y-family DNA polymerases alleviates replication stalling at DNA damage. Ring-shaped processivity clamps play a critical but ill-defined role in mediating exchange between Y-family and replicative polymerases during TLS. By reconstituting TLS at the single-molecule level, we show that the Escherichia coli ß clamp can simultaneously bind the replicative polymerase (Pol) III and the conserved Y-family Pol IV, enabling exchange of the two polymerases and rapid bypass of a Pol IV cognate lesion. Furthermore, we find that a secondary contact between Pol IV and ß limits Pol IV synthesis under normal conditions but facilitates Pol III displacement from the primer terminus following Pol IV induction during the SOS DNA damage response. These results support a role for secondary polymerase clamp interactions in regulating exchange and establishing a polymerase hierarchy.

Remote control of DNA-acting enzymes by varying the Brownian dynamics of a distant DNA end.

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 rates by affecting dynamics leading to transition states. In single-molecule experiments we have found that enzymes that relax DNA torsional stress display rates that depend strongly on how the distant ends of the molecule are constrained; experiments with different-sized particles tethered to the end of 10-kb DNAs reveal enzyme rates inversely correlated with particle drag coefficients. This effect can be understood in terms of the coupling between molecule extension and local molecular stresses: The rate of bead thermal motion controls the rate at which transition states are visited in the middle of a long DNA. Importantly, we have also observed this effect for reactions on unsupercoiled DNA; other enzymes show rates unaffected by bead size. Our results reveal a unique mechanism through which enzyme rates can be controlled by constraints on macromolecular or supramolecular substrates.

Quantitative target engagement of RIPK1 in human whole blood via the cellular thermal shift assay for potential pre-clinical and clinical applications.

The cellular thermal shift assay (CETSA®) is a target engagement method widely used for preclinical characterization of small molecule compounds. CETSA® has been used for semi-quantitative readouts in whole blood with PBMC isolation, and quantitative, plate-based readouts using cell lines. However, there has been no quantitative evaluation of CETSA® in unprocessed human whole blood, which is preferred for clinical applications. Here we report two separate assay formats - Alpha CETSA® and MSD CETSA® - that require less than 100 µL of whole blood per sample without PBMC isolation. We chose RIPK1 as a proof-of-concept target and, by measuring engagement of seven different inhibitors, demonstrate high assay sensitivity and robustness. These quantitative CETSA® platforms enable possible applications in preclinical pharmacokinetic-pharmacodynamic studies, and direct target engagement with small molecules in clinical trials.

Characterizing and Controlling Nanoscale Self-Assembly of Suckerin-12.

Structural proteins such as "suckerins" present promising avenues for fabricating functional materials. Suckerins are a family of naturally occurring block copolymer-type proteins that comprise the sucker ring teeth of cephalopods and are known to self-assemble into supramolecular networks of nanoconfined ß-sheets. Here, we report the characterization and controllable, nanoscale self-assembly of suckerin-12 (S12). We characterize the impacts of salt, pH, and protein concentration on S12 solubility, secondary structure, and self-assembly. In doing so, we identify conditions for fabricating ∼100 nm nanoassemblies (NAs) with narrow size distributions. Finally, by installing a noncanonical amino acid (ncAA) into S12, we demonstrate the assembly of NAs that are covalently conjugated with a hydrophobic fluorophore and the ability to change self-assembly and ß-sheet content by PEGylation. This work presents new insights into the biochemistry of suckerin-12 and demonstrates how ncAAs can be used to expedite and fine-tune the design of protein materials.

Target engagement approaches for pharmacological evaluation in animal models.

The field of chemical biology has introduced several approaches, typically using chemical probes, to measure the direct binding interaction of a small molecule with its biological target in cells. The use of these direct target engagement assays in pharmaceutical development can support mechanism of action hypothesis testing, rank ordering of compounds, and iterative improvements of chemical matter. This Feature Article highlights a newer application of these approaches: the quantification of target engagement in animal models to support late stage preclinical development and the nomination of a drug candidate to clinical trials. Broadly speaking, these efforts can be divided between compounds that covalently and reversibly interact with protein targets; recent examples for both categories are discussed for a range of targets, along with their limitations. New, promising technologies are also highlighted, in addition to the application of target engagement determination to new therapeutic modalities.

Single-molecule imaging reveals multiple pathways for the recruitment of translesion polymerases after DNA damage.

Unrepaired DNA lesions are a potent block to replication, leading to replication fork collapse, double-strand DNA breaks, and cell death. Error-prone polymerases overcome this blockade by synthesizing past DNA lesions in a process called translesion synthesis (TLS), but how TLS polymerases gain access to the DNA template remains poorly understood. In this study, we use particle-tracking PALM to image live Escherichia coli cells containing a functional fusion of the endogenous copy of Pol IV to the photoactivatable fluorescent protein PAmCherry. We find that Pol IV is strongly enriched near sites of replication only upon DNA damage. Surprisingly, we find that the mechanism of Pol IV recruitment is dependent on the type of DNA lesion, and that interactions with proteins other than the processivity factor ß play a role under certain conditions. Collectively, these results suggest that multiple interactions, influenced by lesion identity, recruit Pol IV to sites of DNA damage.