Biology subjects

Houtsmuller, A.

Publications and source records attributed to Houtsmuller, A..

4 recordsLinked to original sources

bioRxiv2024

Regulation of XPG-DNA damage binding dynamics by pre- and post-incision nucleotide excision repair factors and EXO1

The XPG endonuclease plays a crucial role in nucleotide excision repair (NER) and other genome maintenance pathways. Precise regulation of XPG recruitment and activity during DNA repair is essential to avoid erroneous DNA incisions and genomic instability. In this study, we employed live-cell imaging to investigate how XPG function is regulated during NER, focusing on its dynamic interactions with key factors involved in the pre- and post-incision steps. We found that TFIIH and XPA facilitate the recruitment and association of XPG with DNA damage, and that XPG localizes separately from TFIIH to UV-induced lesions. Furthermore, our results show that XPGs dissociation from DNA damage is triggered by its own incision activity as well as by that of XPF. Additionally, the exonuclease EXO1 promotes XPG dissociation, likely by processing incised DNA, even in the absence of XPG-mediated incision. Our findings help to better understand the regulatory mechanisms that control XPG activity during NER and provide important insights into the complex dynamics of the repair process.

genetics↗

bioRxiv2021

Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability

Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA Polymerase II (Pol II), causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions (TBLs). However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR/cas9 screen, we identified elongation factor ELOF1 as an important new factor in the transcription stress response upon DNA damage. We show that ELOF1 has an evolutionary conserved role in Transcription-Coupled Nucleotide Excision Repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair TBLs and resume transcription. Additionally, ELOF1 modulates transcription to protect cells from transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage by two distinct mechanisms.

cell biology↗

bioRxiv2021

Structured illumination microscopy with noise-controlled image reconstructions

Super-resolution structured illumination microscopy (SIM) has become a widely used method for biological imaging. Standard reconstruction algorithms, however, are prone to generate noise-specific artefacts that limit their applicability for lower signal-to-noise data. Here, we present a physically realistic noise model that explains the structured noise artefact and that is used to motivate new complementary reconstruction approaches. True Wiener-filtered SIM optimizes contrast given the available signal-to-noise ratio, flat-noise SIM fully overcomes the structured noise artefact while maintaining resolving power. Both methods eliminate ad-hoc user adjustable reconstruction parameters in favour of physical parameters, enhancing objectivity. The new reconstructions point to a trade-off between contrast and a natural noise appearance. This trade-off can be partly overcome by additional notch filtering, but at the expense of a decrease in signal-to-noise ratio. The benefits of the proposed approaches are demonstrated on focal adhesion and tubulin samples in 2D and 3D, and on nano-fabricated fluorescent test patterns.

biophysics↗

bioRxiv2020

Super-resolution imaging of RAD51 and DMC1 in DNA repair foci reveals dynamic distribution patterns in meiotic prophase

The recombinase RAD51, and its meiosis-specific paralog DMC1 localize at DNA double-strand break (DSB) repair sites in meiotic prophase nuclei. While both proteins are required during meiotic homologous recombination, their spatial organization during meiotic DSB repair is not fully understood. Using super-resolution microscopy on mouse spermatocyte nuclei, we aimed to define their relative position at DSB foci, and how these vary in time. We show that a large fraction of meiotic DSB repair foci (38%) contained a single RAD51 cluster and a single DMC1 cluster (D1R1 configuration) that were partially overlapping (average center-center distance around 70 nm). The majority of the rest of the foci had a similar combination of a major RAD51 and DMC1 cluster, but in combination with additional clusters (D2R1, D1R2, D2R2, or DxRy configuration) at an average distance of around 250 nm. As prophase progressed, less D1R1 and more D2R1 foci were observed, where the RAD51 cluster in the D2R1 foci elongated and gradually oriented towards the distant DMC1 cluster. This correlated with more frequently observed RAD51 bridges between the two DMC1 clusters. D1R2 foci frequency was more constant, and the single DMC1 cluster did not elongate, but was observed more frequently in between the two RAD51 clusters in early stages. D2R2 foci were rare (<10%) and nearest neighbour analyses also did not reveal pair formation between D1R1 foci. In the absence of the transverse filament of the synaptonemal complex (connecting the chromosomal axes of homologs), early configurations were more prominent, and RAD51 elongation occurred only transiently. This in-depth analysis of single cell landscapes of RAD51 and DMC1 accumulation patterns at DSB repair sites at super-resolution thus revealed the variability of foci composition, and defined functional consensus configurations that change over time. AUTHOR SUMMARYMeiosis is a specific type of cell division that is central to sperm and egg formation in sexual reproduction. It forms cells with a single copy of each chromosome, instead of the two copies that are normally present. In meiotic prophase, homologous chromosomes must connect to each other, to be correctly distributed between the daughter cells. This involves the formation and repair of double-strand breaks in the DNA. Here we used super-resolution microscopy to elucidate the localization patterns of two important DNA repair proteins: RAD51 and DMC1. We found that repair sites most often contain a single large cluster of both proteins, with or without one additional smaller cluster of either protein. RAD51 protein clusters displayed lengthening as meiotic prophase progressed. When chromosome pairing was disturbed, we observed changes in the dynamics of protein accumulation patterns, indicating that they actually correspond to certain repair intermediates changing in relative frequency of occurrence. These analyses of single meiotic DNA repair foci reveal the biological variability in protein accumulation patterns, and the localization of RAD51 and DMC1 relative to each other, thereby contributing to our understanding of the molecular basis of meiotic homologous recombination.

developmental biology↗