DNA replication and responses to DNA damage

Research subject 1 Involvement of mammalian NER, TLS and MMR in genome stability and organismal fitness

We have studied the induction of genome instability by low, environmentally relevant, doses of the model DNA damaging agent UV light. Surprisingly, we found that unreplicated DNA lesions could be transferred through mitosis, only collapsing to double-strand DNA breaks during the second S phase after exposure. This resulted in large-scale genomic disarray that caused the emergence of cells carrying a large number of genomic aberrations. We hypothesize that this process of delayed genomic instability is causally involved in chromoplexy, the large-scale genomic rearrangements observed in cancers associated with chronic exposure to low levels of DNA damaging agents (Temviriyanukul et al, submitted).

Mice with a targeted disruption of the core TLS gene Rev1 develop anemia. This phenotype is strongly exacerbated by the simultaneous disruption of the NER gene Xpc, resulting in death by aplastic anemia at 4 months of age. This was associated with replication stress, genomic breaks, DNA damage signaling, senescence, and apoptosis in bone marrow. Surprisingly, the collapse of the Rev1Xpc bone marrow was associated with progressive mitochondrial dysfunction and consequent exacerbation of oxidative stress, and phenotypes of Rev1Xpc hematopoietic stem cells were rescued by culture in the presence or a radical scavenger. These data reveal that, to protect its genomic and functional integrity, the hematopoietic system critically depends on the combined activities of repair and replication of disruptive oxidative nucleotide lesions by NER and TLS, respectively (see publications 1).

We have recently discovered a novel DNA excision repair pathway, called ‘post-TLS repair’ and related to MMR, that excises ‘misincorporations’ by TLS opposite disruptive DNA lesions (see publications 2). This pathway reduces the mutagenicity of TLS while it simultaneously induces checkpoint and apoptotic responses to DNA lesions. Loss of post-TLS repair may provide an explanation for the development of colorectal cancer in Lynch syndrome.

In collaboration with Prof. H. van Bokhoven, Radboud University Medical Center, we have discovered that the congenital neuronal disease Möbius syndrome can be caused by an inborn defect in the TLS gene Rev3. This work underscores the importance of TLS at endogenous DNA lesions for development of the brain (see publications 3).

Research subject 2 Development of diagnostic tools to assess pathogenicity of the common cancer predisposition Lynch syndrome which is caused by an inherited defect in one of the MMR genes

To read more information about the different projects, a lay-over screen will appear with the description of the project. Research subject 2 Development of diagnostic tools to assess pathogenicity of the common cancer predisposition Lynch syndrome which is caused by an inherited defect in one of the MMR genes:
The advent of personalized genomics results in the frequent discovery of genetic variants in MMR genes in individuals suspected of the cancer predisposition Lynch syndrome. Only when the pathogenicity of these variants can be determined, personalized medicine can be applied. We have developed two breakthrough assays to diagnose pathogenicity of variants in MMR genes:

The ‘Complete In Vitro Mismatch Repair Assay’ (CIMRA) that rapidly (one week) measures functionality of MMR gene variants (see publications 4 and References therein). We now have thoroughly calibrated and validated the CIMRA assay and show that a combination of in silico analysis combined with the CIMRA assay is able to assess the pathogenicity of VUS in Msh2 and Mlh1 with high sensitivity and specificity (Drost et al, in preparation).

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In addition to the CIMRA assay we have developed genetic screens to enable the large-scale identification of MMR gene residues, critical for gene function (see publications 5). The resulting ‘Reverse Diagnosis Catalogs’ (RDC) serve to rapidly assess whether a VUS as found in a suspected Lynch syndrome patient causes disease. We now have performed comprehensive screens for the Msh2 and Msh6 genes (manuscript in preparation) and are in the process of performing such screens for the Mlh1 and Pms2 genes.

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Image 4. CIMRA assay of variants in the MMR gene Pms2, identified in individuals suspected of Lynch syndrome. Red bars: suspected pathogenic variants (see publications 4).