Canonical DNA mismatch repair (MMR) excises base-base mismatches to increase the fidelity of DNA replication. Thus, loss of MMR leads to increased spontaneous mutagenesis. MMR genes also are... Show moreCanonical DNA mismatch repair (MMR) excises base-base mismatches to increase the fidelity of DNA replication. Thus, loss of MMR leads to increased spontaneous mutagenesis. MMR genes also are involved in the suppression of mutagenic, and the induction of protective, responses to various types of DNA damage. In this review we describe these non-canonical roles of MMR at different lesion types. Loss of non-canonical MMR gene functions may have important ramifications for the prevention, development and treatment of colorectal cancer associated with inherited MMR gene defects in Lynch syndrome. This graphical review pays tribute to Samuel H. Wilson. Sam not only made seminal contributions to understanding base excision repair, particularly with respect to structure-function relationships in DNA polymerase beta but also, as Editor of DNA Repair, has maintained a high standard of the journal. Show less
DNA encodes the genetic instructions for living organisms. However, damage to the DNA is inevitable, because DNA itself is an unstable molecule and environmental factors such as UV-radiation or X... Show moreDNA encodes the genetic instructions for living organisms. However, damage to the DNA is inevitable, because DNA itself is an unstable molecule and environmental factors such as UV-radiation or X-rays cause damage to the DNA. A certain type of DNA damages can block DNA replication, an essential step before cell can divide. The polymerases that normally replicate DNA are incredibly efficient and virtually flawless on undamaged DNA, but they cannot replicate damaged DNA. In multi-celled organisms, the most important defense mechanism against this is Translesion DNA synthesis (TLS). TLS protects against various negative consequences of damage to the DNA. For this, TLS utilizes specialized TLS polymerases that can replicate damaged DNA.My experiments show that the strong evolutionary conservation of TLS is explained by the dual functions of TLS: guarding replication potential and genome stability. TLS suppresses genomic instability, by preventing conversion of replication blocks to double-stranded DNA breaks (DSBs). Without functional TLS, DSBs arise and result in larger and more harmful mutations. TLS is beneficial for organisms because it supports continuous reproduction and growth. Although DNA damage is always present and unavoidable, TLS guards against the formation of mutations that would otherwise lead to cancer, aging and congenital disease. Show less
Microbial rhodopsins are photosensitive pigments implemented in the growth and adaptation of a large population of microorganisms. These relatively simple, tunable photosystems use a molecule... Show moreMicrobial rhodopsins are photosensitive pigments implemented in the growth and adaptation of a large population of microorganisms. These relatively simple, tunable photosystems use a molecule of retinal as a chromophore to facilitate the conversion of sunlight to chemical energy. Retinal-based phototrophy is believed to sustain the phototrophic balance of various biospheres and has several important biotechnological applications. In this thesis, we propose the use of microbial rhodopsins as an alternative photosystem in a complementary approach towards more efficient use of photons in the solar spectrum. Towards this end, we describe the adaptation of two rhodopsin proton-pumps, namely proteorhodopsin and Gloeobacter rhodopsin, to shift their action spectrum into the near-infrared region. Several red-shifted variants of were generated by utilizing a combination of retinal analogs with specific opsin mutations. We also constructed a novel directed-evolution set-up, which allows us to generate a library of red-shifted mutants with simultaneous screening for spectral shifts and proton-pumping ability. Finally, the impact of a detergent or lipid microenvironment was tested on the various pigments generated in this study. Our results have important prospects in a number of biotechnological fields such as optogenetics, membrane-sensor technology and as a complementary photosystem for oxygenic photosynthesis. Show less
The research described in this thesis has provided new insights in the activation mechanism of class A GPCRs and in particular of adenosine receptors. By a variety of mutagenesis approaches and the... Show moreThe research described in this thesis has provided new insights in the activation mechanism of class A GPCRs and in particular of adenosine receptors. By a variety of mutagenesis approaches and the use of a robust yeast reporter gene system, we identified several regions and amino acid positions that contribute to both agonist responses and constitutive activity of the human adenosine A1 receptor and the human adenosine A2B receptor. These results reveal new and surprising roles of the extracellular loops in the activation mechanism, greatly contributing to our notion of receptor activation. Show less
Het onderzoek beschreven in het proefschrift van B.I. Lindhout heeft zich gericht op het maken van kunstmatige eiwitten die expressie van genen be_nvloeden, zogeheten zinkvinger bevattende artifici... Show moreHet onderzoek beschreven in het proefschrift van B.I. Lindhout heeft zich gericht op het maken van kunstmatige eiwitten die expressie van genen be_nvloeden, zogeheten zinkvinger bevattende artifici_le transcriptiefactoren. Met dit type eiwitten kan worden onderzocht hoe bepaalde processen in een organisme gereguleerd worden, maar ook om op een nieuwe manier eigenschappen van een organisme te veranderen. Een speciaal type DNA-bindende zinkvingers vormt een onderdeel van deze eiwitten. Het proefschrift beschrijft experimenten die zijn gericht op de verdere uitwerking van deze techniek, vooral voor het plantenbiologisch onderzoek. Er wordt aangetoond dat zinkvinger transcriptiefactoren inderdaad goed gebruikt kunnen worden om een specifiek gen te reguleren, maar ook hoe een grote verzameling daarvan gebruikt kan worden om te zoeken naar verborgen genetische eigenschappen. Deze speciale toepassing houdt in dat specifieke kenmerken die een plant in principe van zichzelf zou kunnen hebben, maar die ontbreken omdat het eigen genetische materiaal van de plant onvoldoende tot uitdrukking komt, plotseling zichtbaar kunnen worden. Zo is gebleken dat introductie van een bepaalde transcriptiefactor de plant in staat stelt zijn DNA veel nauwkeuriger te repareren. Eveneens is beschreven hoe lichtgevende zinkvingers gebruikt kunnen worden om __live__ naar het genetische materiaal van planten- en muizencellen te kunnen kijken. Show less
In this thesis the role of DNA mismatch repair (MMR) in the cellular response to several genotoxic agents is described. We show that MMR plays an important role in the protection against UVC... Show moreIn this thesis the role of DNA mismatch repair (MMR) in the cellular response to several genotoxic agents is described. We show that MMR plays an important role in the protection against UVC-induced mutagenesis in mouse embryonic stem (ES) cells. UVC was shown to induce six times more mutations in mouse ES cells deficient for the mismatch recognition dimer MutSalpha compared to wild type cells. The Hprt mutational spectrum of UVC-induced mutations was similar in MutSalpha-proficient and MutSalpha-deficient mouse ES cells. We subsequently tried to gain insight into the mechanism by which MMR mediates protection against UVC-induced mutagenesis. We found that UVC induces a late S/G2-phase arrest which partially depends on the presence of MutSalpha. The MutSalpha-dependent late S/G2-phase arrest coincided with the appearance of phosphorylated Chk-1 of which the levels were higher in MutSalpha-proficient cells compared to MutSalpha-deficient cells. Importantly, abolishment of the UVC-induced late S/G2-phase arrest in both MutSalpha-proficient and MutSalpha-deficient cells did not result in a smaller difference in mutation induction between both genotypes after UVC treatment. We propose that MMR removes mismatches from UVC-induced compound lesions and that the MutSalpha-induced late S/G2-phase arrest is the result of the appearance of DNA single stranded regions arising during this process of MMR. 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
