Natural products provide a rich source of potential antimicrobials for treating infectious diseases for which drug resistance has emerged. Foremost among these diseases is tuberculosis. Assessment... Show moreNatural products provide a rich source of potential antimicrobials for treating infectious diseases for which drug resistance has emerged. Foremost among these diseases is tuberculosis. Assessment of the antimycobacterial activity of nargenicin, a natural product that targets the replicative DNA polymerase of Staphylococcus aureus, revealed that it is a bactericidal genotoxin that induces a DNA damage response in Mycobacterium tuberculosis (Mtb) and inhibits growth by blocking the replicative DNA polymerase, DnaE1. Cryo-electron microscopy revealed that binding of nargenicin to Mtb DnaE1 requires the DNA substrate such that nargenicin is wedged between the terminal base pair and the polymerase and occupies the position of both the incoming nucleotide and templating base. Comparative analysis across three bacterial species suggests that the activity of nargenicin is partly attributable to the DNA binding affinity of the replicative polymerase. This work has laid the foundation for target-led drug discovery efforts focused on Mtb DnaE1. Show less
Multidrug resistance is a worldwide problem that is an increasing threat to global health. Therefore, the development of new antibiotics that inhibit novel targets is of great urgency. Some of the... Show moreMultidrug resistance is a worldwide problem that is an increasing threat to global health. Therefore, the development of new antibiotics that inhibit novel targets is of great urgency. Some of the most successful antibiotics inhibit RNA transcription, RNA translation, and DNA replication. Transcription and translation are inhibited by directly targeting the RNA polymerase or ribosome, respectively. DNA replication, in contrast, is inhibited indirectly through targeting of DNA gyrases, and there are currently no antibiotics that inhibit DNA replication by directly targeting the replisome. This contrasts with antiviral therapies where the viral replicases are extensively targeted. In the last two decades there has been a steady increase in the number of compounds that target the bacterial replisome. In particular a variety of inhibitors of the bacterial replicative polymerases PolC and DnaE have been described, with one of the DNA polymerase inhibitors entering clinical trials for the first time. In this review we will discuss past and current work on inhibition of DNA replication, and the potential of bacterial DNA polymerase inhibitors in particular as attractive targets for a new generation of antibiotics. Show less
Elsayed, F.A.; Tops, C.M.J.; Nielsen, M.; Ruano, I.; Vasen, H.F.A.; Morreau, H.; ... ; Wezel, T. van 2019
Background Germline mutations affecting the exonuclease domains of POLE and POLD1 predispose to colorectal adenomas and carcinoma. Here, we aimed to screen the exonuclease domains to find the... Show moreBackground Germline mutations affecting the exonuclease domains of POLE and POLD1 predispose to colorectal adenomas and carcinoma. Here, we aimed to screen the exonuclease domains to find the genetic causes of multiple colorectal polyps in unexplained cases. Methods Using a custom next-generation sequencing panel, we sequenced the exonuclease domains of POLE and POLD1 in 332 index patients diagnosed with multiple colorectal polyps without germline alteration in colorectal polyposis predisposing genes. Results We identified two variants of unknown significance. One germline POLD1 c.961G>A, p.(Gly321Ser) variant was found in two cases. The first patient was diagnosed with multiple polyps at age 35 and colorectal cancer (CRC) at age 37, with no known family history of CRC. The second patient was diagnosed with CRC at age 44 and cumulatively developed multiple polyps; this patient had two sisters with endometrial cancer who did not carry the variant. Furthermore, we identified a novel POLD1 c.955 T>G, p.(Cys319Gly) variant in a patient diagnosed with multiple colorectal adenomas at age 40. Co-segregation analysis showed that one sister who cumulatively developed multiple adenomas from age 34, and another sister who developed CRC at age 38 did not carry the variant. We did not identify pathogenic variants in POLE and POLD1. Conclusion This study confirms the low frequency of causal variants in these genes in the predisposition for multiple colorectal polyps, and also establishes that these genes are a rare cause of the disease. Show less
The replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45–MCM–GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The... Show moreThe replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45–MCM–GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The mechanism for coupling DNA unwinding with synthesis is starting to be elucidated, however the architecture and dynamics of the replication fork remain only partially understood, preventing a molecular understanding of chromosome replication. To address this issue, we conducted a systematic single-particle EM study on multiple permutations of the reconstituted CMG–Pol epsilon assembly. Pol epsilon contains two flexibly tethered lobes. The noncatalytic lobe is anchored to the motor of the helicase, whereas the polymerization domain extends toward the side of the helicase. We observe two alternate configurations of the DNA synthesis domain in the CMG-bound Pol epsilon. We propose that this conformational switch might control DNA template engagement and release, modulating replisome progression. Show less
The overall aim of this thesis is to contribute to the engineering of more selective and effective oncolytic Adenovirus (Ad) vectors. Two general approaches are taken for this purpose: (i) genetic... Show moreThe overall aim of this thesis is to contribute to the engineering of more selective and effective oncolytic Adenovirus (Ad) vectors. Two general approaches are taken for this purpose: (i) genetic capsid modification to achieve Ad retargeting (Chapters 2 to 4), and (ii) directed evolution to improve the cytolytic potency of Ad (Chapter 5). In order to provide some context for these approaches, Chapter 1, part II gives a brief background on Ad biology and vectorology. Further, in Chapter 1, part III, a broad overview is provided of the ways that evolution-based engineering has previously been used to generate or improve viral vectors. Chapters 2 and 3 focus on the modification of the minor Ad capsid protein IX (pIX). pIX is present on the faces of the Ad capsid icosahedron, functioning as __cement__ between the much larger hexon proteins. Previously, the C-terminus of pIX proved serviceable as an anchor for the genetic capsid incorporation of targeting ligands and other heterologous moieties. In Chapter 2, a new system is described that allows for the rapid functional testing of new pIX-ligand fusion proteins. In this system, lentiviral vectors are used to generate cells stably expressing the pIX variant of interest. Large-scale infection on such cells with a pIX-deleted Ad vector subsequently yields an Ad vector preparation phenotypically pseudotyped with the new pIX variant. This system thus allows rapid analysis of new pIX-ligand fusions in the context of the Ad capsid without having to genetically modify the Ad genome. In Chapter 3, the lentiviral vector-based pIX-pseudotyping system is put to use for the analysis of a new pIX fusion protein harboring a single-chain T-cell receptor (scTCR) as a targeting ligand. The concerning scTCR was directed against the intracellular cancer-testis antigen melanoma-associated antigen-A1. Importantly, this chimeric pIX molecule proved to be efficiently incorporated into the Ad capsid. Moreover, Ad transduction studies showed evidence of the capsid-displayed scTCR to mediate a degree of specific target cell transduction via the cognate peptide-MHC complex. Analogously as done for pIX, Chapter 4 describes a phenotypical pseudotyping approach for fiber. The Ad-encoded fiber protein is present as a trimeric rod-like structure that extends from the vertices of the Ad capsid icosahedron. Its outward-facing, C-terminal __knob__ domain is responsible for binding the Coxsackie and adenovirus receptor (CAR), Ad__s in vitro primary cell surface attachment protein. With its prominent role in native receptor binding, the Ad fiber is logically subject to many capsid modification strategies that aim at altering Ad tropism. Thus to facilitate expedited testing of new fiber variants, a lentiviral vector-based, fiber-pseudotyping system was set up. This involved optimization of the fiber (variant) expression cassettes by inclusion of the tripartite leader sequence of Ad__s major late transcription unit. A second objective of this study was to functionally assess a new chimeric fiber harboring a tumor antigen-directed single-chain variable fragment (scFv) antibody. Although this fiber variant showed some degree of target binding and formed stable trimers, it displayed problems regarding capsid incorporation ability, functionality within the capsid, and folding of its scFv constituent. Thus, this particular fiber proved not suitable for Ad retargeting. Finally, Chapter 5 describes the development and validation of a novel evolution-based engineering approach for Ad. To date, most Ad-based vectors have been generated through molecular design. Although this rational tailoring of Ad has led to significant vector improvements, it is often still hampered by our limited understanding of the intricate viral function-structure relationships. Therefore, __random__ virus engineering strategies (see Chapter 1, part III) may be a useful alternative or complementary approach for the generation of new or improved viral vectors. In this regard, the high mutation rates of RNA viruses have proven readily exploitable in adaptation studies to achieve vectorological goals. Thus, it was hypothesized that a mutator Ad polymerase-based, __accelerated evolution__ procedure would likewise be of use for Ad vector engineering. To develop such a system, the intrinsic mutation rate of Ad replication was sought to be increased by modification of the Ad-encoded DNA polymerase (Ad pol). This was done by mutation of residues within regions putatively important for nucleotide selection or proofreading. A mutation-accumulation and deep sequencing strategy was subsequently used to identify any mutators among the Ad pol mutants. Finally, the mutator polymerase-based directed evolution approach was validated by conducting an evolution procedure aimed at increasing Ad__s oncolytic potency, and by subsequent characterization of resultant bioselected virus populations and isolated clones. Show less
