http://repub.eur.nl/res/aut/4217/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/30196/ 2008-12-01T00:00:00Z Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) η and ι are usually uniformly distributed through the nucleus but accumulate in replication foci during S phase. DNA-damaging treatments result in an increase in S phase cells containing polymerase foci. Using photobleaching techniques, we show that polη is highly mobile in human fibroblasts. Even when localized in replication foci, it is only transiently immobilized. Although ubiquitination of proliferating cell nuclear antigen (PCNA) is not required for the localization of polη in foci, it results in an increased residence time in foci. polι is even more mobile than polη, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both polη and polι diffuse through the cell but that they are transiently immobilized for ∼150 ms, with a larger proportion of polη than polι immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of polη but not polι. Our data are consistent with a model in which the polymerases are transiently probing the DNA/chromatin. When DNA is exposed at replication forks, the polymerase residence times increase, and this is further facilitated by the ubiquitination of PCNA. http://repub.eur.nl/res/pub/12292/ 2008-05-14T00:00:00Z The integrity of the genome, carrier of the blueprint for each organism, is under constant attack from environmental as well as endogenous DNA damaging agents. An agent with substantial impact on our DNA is the UV-fraction of sunlight. It inflicts bulky DNA lesions, which can interfere with vital cellular functions, such as DNA replication and transcription of genes, by simply blocking these processes. This can lead to cell death or to mutations, which in higher organisms can in turn lead to inborn diseases and cancer. To counteract these deleterious effects, the specialized multi-component nucleotide excision repair (NER) machinery has evolved which removes and replaces the damaged oligonucleotide in a multi-step process. Defects in this repair pathway are the underlying cause for the inherited UV-sensitive diseases xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. In association with these disorders it has been demonstrated that repair deficiencies do not only lead to mutations and cancer, but also to developmental difficulties, (neuronal) degeneration and premature ageing. In rapidly dividing cells, not all DNA lesions can be repaired by the time a new DNA replication round has begun, even if the corresponding repair pathway is fully functional. Therefore, cells have also evolved specialized lesion-bypass polymerases. Translesion polymerases are capable of resolving DNA polymerase complexes stalled at DNA damage sites by inserting a nucleotide opposite an altered base, enabling the cell to continue to replicate. Importantly, polymerase eta can insert the correct nucleotide opposite UVinduced pyrimidine-pyrimidine adducts such as cyclobutane pyrimidine dimers (CPD), enabling mutation-free replication of DNA past these UV lesions. Without a functioning polymerase eta, unrepaired UV injuries blocking the replication process can only be resolved by mutational translesion synthesis performed by other lesion bypass polymerases. This leads to a highly elevated risk of skin cancer in patients exhibiting a special form of xeroderma pigmentosum due to lack of polymerase eta. http://repub.eur.nl/res/pub/3218/ 2004-07-05T00:00:00Z The Cockayne syndrome B (CSB) protein is essential for transcription-coupled DNA repair (TCR), which is dependent on RNA polymerase II elongation. TCR is required to quickly remove the cytotoxic transcription-blocking DNA lesions. Functional GFP-tagged CSB, expressed at physiological levels, was homogeneously dispersed throughout the nucleoplasm in addition to bright nuclear foci and nucleolar accumulation. Photobleaching studies showed that GFP-CSB, as part of a high molecular weight complex, transiently interacts with the transcription machinery. Upon (DNA damage-induced) transcription arrest CSB binding these interactions are prolonged, most likely reflecting actual engagement of CSB in TCR. These findings are consistent with a model in which CSB monitors progression of transcription by regularly probing elongation complexes and becomes more tightly associated to these complexes when TCR is active. http://repub.eur.nl/res/pub/8360/ 2004-01-01T00:00:00Z The Cockayne syndrome B (CSB) protein is essential for transcription-coupled DNA repair (TCR), which is dependent on RNA polymerase II elongation. TCR is required to quickly remove the cytotoxic transcription-blocking DNA lesions. Functional GFP-tagged CSB, expressed at physiological levels, was homogeneously dispersed throughout the nucleoplasm in addition to bright nuclear foci and nucleolar accumulation. Photobleaching studies showed that GFP-CSB, as part of a high molecular weight complex, transiently interacts with the transcription machinery. Upon (DNA damage-induced) transcription arrest CSB binding these interactions are prolonged, most likely reflecting actual engagement of CSB in TCR. These findings are consistent with a model in which CSB monitors progression of transcription by regularly probing elongation complexes and becomes more tightly associated to these complexes when TCR is active.