Biology subjects

Tijsterman, M.

Publications and source records attributed to Tijsterman, M..

8 recordsLinked to original sources

bioRxiv2026

A direct RPA-Polθ interaction licenses theta-mediated end joining

DNA double-strand break repair often involves end resection, yet how resected ends are directed toward distinct repair pathways remains poorly understood. Here, we identify replication protein A (RPA) as an unexpected essential factor for cellular polymerase theta-mediated end joining (TMEJ). Using MUSIC-MAP, a newly developed platform linking endogenous sequence variants to DNA repair outcomes, we map a discrete functional interface between RPA2 and polymerase theta (Pol{theta}). Disrupting this interface in RPA2 abolishes TMEJ without impairing cell viability or homologous recombination. Mutations in the complementary Pol{theta} surface likewise impair TMEJ, whereas reciprocal charge-reversal mutations in RPA2 and Pol{theta} restore TMEJ, providing genetic evidence for a direct physical interaction. Structural modeling suggests that the Pol{theta}-binding surface becomes accessible at RPA-coated ssDNA termini, positioning RPA as a docking platform for Pol{theta} engagement. These findings reveal how RPA, previously considered a barrier to TMEJ, instead functions as a molecular platform that translates the organization of ssDNA into selective enzyme engagement, enabling mutagenic repair without compromising homologous recombination.

molecular biology↗

bioRxiv2026

CIP2A tetramerization is required for mitotic DNA repair

DNA lesions that persist in mitosis threaten genome stability. These lesions recruit TOPBP1-CIP2A, a complex crucial to tether and process damaged DNA on mitotic chromosomes. Importantly, CIP2A is synthetic lethal in BRCA1/2 mutant cancer cells. However, mechanistic insight into the function of CIP2A is lacking. Here we provide first structural insights into full-length CIP2A and reveal how CIP2A dimers, 46 nm in length, assemble via conserved C-terminal motifs into dumbbell-like tetramers. DNA repair signatures demonstrate that CIP2A tetramerization is crucial for mitotic DNA repair by polymerase theta (POLQ). Tetramerization of CIP2A is needed to recruit POLQ to mitotic DNA lesions and to prevent micronucleation upon replication-born DNA lesions. Tetramerization-defective CIP2A mutants specifically decrease survival of BRCA1/2 mutant cancer cells in a dominant negative manner. Combined, we demonstrate that CIP2A tetramers are crucial components of a mitotic DNA-tethering and repair scaffold, which processes replication-mediated DNA lesions that persist in mitosis.

cell biology↗

bioRxiv2025

Signatures in CRISPR Mutational Spectra Reveal Role and Interplay of Genes in DNA Repair

MotivationUnderstanding double-strand break (DSB) repair and its disruption is key to decipher genomic instability driving diseases such as cancer and reveal therapeutic avenues. Numerous genes have been linked to DSB repair for the first time in recent genome-wide perturbation studies assessing effects on mutational outcomes. However, the functional roles of most such genes remain poorly understood. Evidence from other studies shows that related genes similarly modulate the frequency of specific mutational outcomes following DSB repair, but analysis has largely ignored the multiplicity of gene functions and cross-talk between pathways. Here, we infer functional roles for candidate genes based on knockout mutational spectra by identifying mutational signatures shared with known genes and then mapping them to DSB repair functions. Signatures are identified using non-negative matrix factorization (NMF) to reflect functional multiplicity and cross-talk. ResultsWe generated mutational spectra for mouse embryonic stem (mES) cells at three target sites across CRISPR knockouts of 307 known and 459 candidate DSB repair genes. Analysis using NMF revealed four mutational signatures associated with homology-directed repair (HDR), microhomology-mediated end-joining (MMEJ), and the initiation and ligation components of non-homologous end-joining (NHEJ). Signatures suggested that candidate genes Dbr1 and Hnrnpk could influence MMEJ and Fanconi anaemia (FA), and that loss of core NHEJ components (e.g. MRN complex or Ku proteins) could shift repair preference towards Ku-independent NHEJ. These findings demonstrate the utility of NMF for characterizing the contribution of genes to repair pathways and provide a foundation to discover new gene functions in DSB repair. Availabilitygithub.com/joanagoncalveslab/MuSpectraNMF.

bioinformatics↗

bioRxiv2025

Accurate characterization of CRISPR-Cas9 genome editing outcomes and mosaicism with near-perfect long reads

BackgroundGenetic mosaicism is a consequence of CRISPR-Cas9 genome editing that is difficult to study, especially when it involves structural variants occurring at low frequency. A comprehensive analysis of mosaicism requires deep and unbiased sequencing of the target loci, with accurate single molecule reads. Here we performed amplification-free PureTarget PacBio sequencing to investigate CRISPR-Cas9 outcomes at on-target and off-target sites in germline edited zebrafish and their offspring. ResultsThirty samples from pooled larvae and individual zebrafish were successfully sequenced, resulting in >1100x average target coverage. The PacBio reads reached an exceptional accuracy (QV39) over the target regions, with every read originating from a unique DNA molecule. The two haplotypes of the target loci displayed a balanced depth of coverage, while long-range PCR of the same samples resulted in skewed data. Further analysis of the PureTarget data revealed widespread genetic mosaicism in individual founder (F0) fish, with up to 18 distinct on-target events and 11 off-target events present in a single adult founder. Several CRISPR-Cas9 editing outcomes, including large structural variants and off-target mutations, were inherited to the F1 generation. Notably, as many as seven unique editing events were found among F1 juvenile offspring from a single founder pair, thereby confirming the presence of genetic mosaicism in germ cells of founder zebrafish. This implies that some consequences of CRISPR-Cas9 editing may emerge only in the second generation. We also analyzed DNA methylation but did not observe altered 5mC CpG signals in genome edited samples. ConclusionsPureTarget enables efficient, accurate and unbiased analysis of the full spectrum of genetic mosaicism in a sample. The potential risks of introducing germ cell mosaicism in founder individuals should be carefully evaluated when designing germline genome editing experiments.

genomics↗

bioRxiv2025

Mutational signature catalogue (MUSIC) of DNA double-strand break repair

Genome alterations arise from inaccurate DNA repair, accumulating into distinct mutational signatures. Here, we investigate the role of every genomically encoded gene in double-strand break (DSB) repair by generating high-resolution outcome profiles following gene knockouts. Using a CRISPR/Cas9-based, massive-parallel bulk library approach, we construct a comprehensive, user-explorable mutational signature catalogue (MUSIC), mapping the full repertoire of DSB repair factors. Our analysis identifies and validates gene clusters - including nearly all known and several novel genes - linked to non-homologous end-joining, 53BP1 sub-pathways, homology-directed repair, and polymerase Theta (POLQ)-mediated end-joining. By focusing on pathway-specific repair outcomes, we uncover a previously unrecognized role for the WRN helicase in suppressing inverted templated insertions, a poorly understood POLQ-associated mutational signature also found in human disease alleles. Furthermore, in-depth analysis of MUSICs scar features reveals unexpected distinctions among genes within the same pathway, providing mechanistic insight and opening multiple new avenues for investigation into chromosomal break repair.

genetics↗

bioRxiv2025

MUSICiAn: Genome-wide Identification of Genes Involved in DNA Repair via Control-Free Mutational Spectra Analysis

MotivationUnderstanding the factors involved in DNA double-strand break (DSB) repair is crucial for the development of targeted anti-cancer therapies, yet the roles of many genes remain unclear. Recent studies show that perturbations of certain genes can alter the distribution of sequence-specific mutations left behind after DSB repair. This suggests that genome-wide screening could reveal novel DSB repair factors by identifying genes whose perturbation causes the mutational distribution spectra observed at a given DSB site to deviate significantly from the wild-type. However, designing proper controls for a genome-wide perturbation screen could be challenging. We explore the idea that a genome-wide screen might allow us to forgo the use of traditional non-targeting controls by reframing the analysis as an outlier detection problem, assuming that most genes have minimal influence on DSB repair. ResultsWe propose MUSICiAn (Mutational Signature Catalogue Analysis), a compositional data analysis method that ranks gene perturbation-specific mutational spectra without controls by measuring deviations from the central tendency in the distributions of all spectra. We show that MUSICiAn can effectively estimate pseudo-controls for the existing Repair-seq dataset, screening 476 genes and 60 non-targeting controls. We further apply MUSICiAn to a genome-wide dataset profiling mutational outcomes induced by CRISPR-Cas9 at three target sites across cells with individual perturbations of 18,406 genes. MUSICiAn successfully recovers known genes, highlights the spliceosome as a lesser-appreciated player in DSB repair, and reveals candidates for further investigation. Availabilitygithub.com/joanagoncalveslab/MUSICiAn.

bioinformatics↗

bioRxiv2022

SIQ: easy quantitative measurement of mutation profiles in sequencing data

Establishing mutational outcomes after genome editing is of increasing importance with the advent of highly efficient genome-targeting tools. Next-generation sequencing (NGS) has become a vital method to investigate the extent of mutagenesis at specific target sites. Thus, robust and simple-to-use software that enables researchers to retrieve mutation profiles from NGS data is needed. Here, we present Sequence Interrogation and Quantification (SIQ), a tool that can analyse sequence data of any targeted experiment (e.g. CRISPR, I-SceI, TALENs) with a focus on event classification such as deletions, single-nucleotide variations, (templated) insertions and tandem duplications. SIQ results can be directly analysed and visualized via SIQPlotteR, an interactive web tool that we made freely available. Using novel and insightful tornado plot visualizations as outputs we illustrate that SIQ readily identifies differences in mutational signatures obtained from various DNA-repair deficient genetic backgrounds. SIQ greatly facilitates the interpretation of complex sequence data by establishing mutational profiles at specific loci and is, to our knowledge, the first tool that can analyse Sanger sequence data as well as short and long-read NGS data (e.g. Illumina and PacBio).

bioinformatics↗

bioRxiv2019

Low Dose Ionizing Radiation Strongly Stimulates Insertional Mutagenesis in a γH2AX Dependent Manner

Extrachromosomal DNA can integrate into the genome with no sequence specificity producing an insertional mutation. This process, which is referred to as random integration (RI), requires a double stranded break (DSB) in the genome. Inducing DSBs by various means, including ionizing radiation, increases the frequency of integration. Here we report that non-lethal physiologically relevant doses of ionizing radiation (10-100 mGy), within the range produced by medical imaging equipment, stimulate RI of transfected and viral episomal DNA in human and mouse cells with an extremely high efficiency. Genetic analysis of stimulated RI (S-RI) revealed that it is distinct from the background RI, requires histone H2AX S139 phosphorylation ({gamma}H2AX) and is not reduced by DNA polymerase {theta} (Polq) inactivation. S-RI efficiency was unaffected by the main DSB repair pathway (homologous recombination and non-homologous end joining) disruptions, but double deficiency in MDC1 and 53BP1 phenocopies {gamma}H2AX inactivation. The robust responsiveness of S-RI to physiological amounts of DSBs has implications for radiation risk assessment and can be exploited for extremely sensitive, macroscopic and direct detection of DSB-induced mutations.

molecular biology↗