This thesis investigates the characteristics and the mechanisms of DNA replicative proteins through the use of biochemistry, single molecule light mycroscopy and Cryo-EM.
Introduction contains a general overview of the research topics discussed in this thesis.Chapter 1 addresses a fundamental question in DNA mismatch repair, which is how ATP binding and hydrolysis... Show moreIntroduction contains a general overview of the research topics discussed in this thesis.Chapter 1 addresses a fundamental question in DNA mismatch repair, which is how ATP binding and hydrolysis drive the conformational changes in MutS thatare needed for the mismatch repair cascade.Chapter 2 focuses on the final stages of the DNA mismatch repair pathway, which are the resection and subsequent resynthesis of the mismatch containing strand.Chapter 3 presents an example of how DNA polymerases can be targeted for the development of novel antibiotics against Mycobacterium tuberculosis (Mtb).Chapter 4 describes a new instrument named the Puffalot, developed for the preparation of cryo-EM grids, which aims to improve the reliability of cryo-EM sample preparation.Discussion provides a summary of the scientific findings described in this thesis in light of the published literature as well as an overview of the future directions and perspectives. Show less
DNA interstrand crosslinks (ICLs) are cytotoxic lesions that threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI... Show moreDNA interstrand crosslinks (ICLs) are cytotoxic lesions that threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI-FANCD2 (ID2) marking the activation step that triggers incisions on DNA to unhook the ICL. Restoration of intact DNA requires the coordinated actions of polymerase zeta (Pol zeta)-mediated translesion synthesis (TLS) and homologous recombination (HR). While the proteins mediating FA pathway activation have been well characterized, the effectors regulating repair pathway choice to promote error-free ICL resolution remain poorly defined. Here, we uncover an indispensable role of SCAI in ensuring error-free ICL repair upon activation of the FA pathway. We show that SCAI forms a complex with Pol zeta and localizes to ICLs during DNA replication. SCAI-deficient cells are exquisitely sensitive to ICL-inducing drugs and display major hallmarks of FA gene inactivation. In the absence of SCAI, HR-mediated ICL repair is defective, and breaks are instead re-ligated by polymerase theta-dependent microhomology-mediated end-joining, generating deletions spanning the ICL site and radial chromosomes. Our work establishes SCAI as an integral FA pathway component, acting at the interface between TLS and HR to promote error-free ICL repair. Show less
The studies presented in this thesis aimed to better understand specific C. difficile physiological processes: genome maintenance, DNA replication, and toxin regulation. As the available tools to... Show moreThe studies presented in this thesis aimed to better understand specific C. difficile physiological processes: genome maintenance, DNA replication, and toxin regulation. As the available tools to investigate C difficile were insufficient, a secondary goal was to develop novel methods for C. difficile studies. We developed a luciferase toolkit for use in C. difficile, not only for gene expression analysis, but also for protein-protein interaction studies in vivo, secretion of proteins or the analysis of complexes present at the cell surface. We investigated fluorescence microscopy for C. difficile, by comparing different fluorescent systems previously described, and introduced the use of the fluorophore HaloTag. Additionally, we analyzed C. difficile intrinsic fluorescence and report the potential limitations for live-cell microscopy. The development of the tools enabled the assessment of the localization of the TcdC C-terminus and the characterization of the HupA protein. Finally, we provide new insights into the chromosome remodelling and DNA replication by studying DnaA-dependent unwinding and characterizing the histone-like protein HupA. In the discussion, we present the implications of our findings and provide perspectives for further research on TcdC-mediated toxin regulation and chromosome dynamics, specifically during DNA replication. 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
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 Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have... Show moreThe Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3′ single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase. Show less
The work described in this thesis deals with characterization of DNA binding by the BRCT domain of the large subunit of RFC. Replication Factor C (RFC) is a five protein complex involved in... Show moreThe work described in this thesis deals with characterization of DNA binding by the BRCT domain of the large subunit of RFC. Replication Factor C (RFC) is a five protein complex involved in initiating and regulating new DNA synthesis. The first half of the thesis describes region of the RFC and structural determinants of DNA required for productive protein-DNA interaction. The second half describes three-dimensional structure determination of the protein-DNA complex, which consists of the BRCT region of the RFC and doubled stranded DNA. The resulting structure based on the data from NMR and mutagenesis reveals structural conservations of few amino acids among the members of BRCT domain superfamily, which are known to bind either phosphorylated peptide or DNA. The work may help us to identify other potential DNA binding BRCT domains. Show less
