Biology subjects

Knipscheer, P.

Publications and source records attributed to Knipscheer, P..

7 recordsLinked to original sources

bioRxiv2025

Tissue-specific mutagenesis from endogenous guanine damageis suppressed by Polκ and DNA repair

Knowledge of mutational patterns has expanded significantly, but a persistent challenge is to link these complex patterns to the underlying molecular mechanism or source of DNA damage, especially when that damage is endogenous and not driven by environmental mutagens. Technological advances now allow us to catalogue mutation across tissues or even closely related cell types, but the results are largely descriptive until we identify the endogenous sources of mutation and how they differ across tissues. Here, we combine mouse genetics, advanced sequencing, biochemistry and mass spectrometry to provide a detailed mechanistic understanding of endogenous mutagenesis. We reveal that endogenous guanine adducts are significant drivers of tissue-specific mutagenesis, while the interwoven actions of DNA polymerase Pol{kappa} and DNA repair mechanisms are pivotal in mitigating mutagenesis. For the first time, we use untargeted DNA adductomics to characterize new sources of endogenous DNA damage. Our novel approach to understanding endogenous mutational landscapes reveals previously unseen mutational processes and points to vast potential for new discoveries.

molecular biology↗

bioRxiv2023

BRCA2-HSF2BP Oligomeric Ring Disassembly by BRME1 Promotes Homologous Recombination

In meiotic homologous recombination (HR), BRCA2 facilitates loading of the recombinases RAD51 and DMC1 at the sites of double-strand breaks. The HSF2BP-BRME1 complex interacts with BRCA2 to support its function in meiotic HR. In somatic cancer cells ectopically producing HSF2BP, DNA damage can trigger HSF2BP-dependent degradation of BRCA2, which prevents HR. Here we show that, upon binding to BRCA2, HSF2BP assembles into a large ring-shaped 24-mer consisting of three interlocked octameric rings. Addition of BRME1 leads to dissociation of this ring structure, and cancels the disruptive effect of HSF2BP on cancer cell resistance to DNA damage. It also prevents BRCA2 degradation during inter-strand DNA crosslink repair in Xenopus egg extracts. We propose that the control of HSF2BP-BRCA2 oligomerization by BRME1 ensures timely assembly of the ring complex that concentrates BRCA2 and controls its turnover, thus promoting meiotic HR.

biochemistry↗

bioRxiv2023

RNA transcripts suppress G-quadruplex structures through G-loop formation

The eukaryotic genome contains numerous guanine-rich sequences that regulate DNA replication and transcription by folding into non-canonical structures called G-quadruplexes (G4s)1. Compromised G4 resolution causes genomic instability and diseases linked to cancer susceptibility and aging2. However, how G4s are resolved is poorly understood. G4 structures are found in genomic regions containing DNA:RNA hybrids, also referred to as R-loops3, 4. When an R-loop and G4 structure form exactly opposite to each other, a G-loop structure is formed. G-loops have been observed directly in prokaryotes5 but their function is unknown. Using the Xenopus egg extract system, we show that G-loop structures act in suppressing mutagenic G4 structures thereby preventing genomic instability. Mechanistically, the RAD51 recombinase binds to the G4-opposing strand and promotes hybridization of RNA transcripts supplied by the hnRNPA1 ribonucleoprotein complex independently of ongoing transcription. The resulting G-loop then induces FANCI-FANCD2 monoubiquitination that triggers site-specific incision of the hybrid strand by the SLX4-XPF-ERCC1 complex. G-loop incision requires prior G4 unwinding by the DHX36 and FANCJ helicases and allows immediate DNA synthesis past the G4 motif. This resynthesis step impacts local chromatin states. This work establishes a G-loop-dependent mechanism that prevents mutagenic consequences of both G4 and R-loop structures.

biochemistry↗

bioRxiv2021

Sister chromatid exchanges induced by perturbed replication are formed independently of homologous recombination factors

Sister chromatid exchanges (SCEs) are products of joint DNA molecule resolution, and are considered to form through homologous recombination (HR). Indeed, upon generation of irradiation-induced DNA breaks, SCE induction was compromised in cells deficient for canonical HR factors BRCA1, BRCA2 and RAD51. Contrarily, replication-blocking agents, including PARP inhibitors, induced SCEs independently of BRCA1, BRCA2 and RAD51. PARP inhibitor-induced SCEs were enriched at common fragile sites (CFSs), and were accompanied by post-replicative single-stranded DNA (ssDNA) gaps. Moreover, PARP inhibitor-induced replication lesions were transmitted into mitosis, suggesting that SCEs originate from mitotic processing of under-replicated DNA. We found that DNA polymerase theta (POLQ) was recruited to mitotic DNA lesions, and loss of POLQ resulted in reduced SCE numbers and severe chromosome fragmentation upon PARP inhibition in HR-deficient cells. Combined, our data show that PARP inhibition generates under-replicated DNA, which is transferred into mitosis and processed into SCEs, independently of canonical HR factors.

cancer biology↗

bioRxiv2021

Single-embryo phosphoproteomics reveals the importance of intrinsic disorder in cell cycle dynamics

Switch-like cyclin-dependent kinase (CDK)-1 activation is thought to underlie the abruptness of mitotic onset, but how CDKs can simultaneously phosphorylate many diverse substrates is unknown, and direct evidence for such phosphorylation dynamics in vivo is lacking. Here, we analysed protein phosphorylation states in single Xenopus embryos throughout synchronous cell cycles. Over a thousand phosphosites were dynamic in vivo, and assignment of cell cycle phases using egg extracts revealed hundreds of S-phase phosphorylations. Targeted phosphoproteomics in single embryos showed switch-like mitotic phosphorylation of diverse protein complexes. The majority of cell cycle-regulated phosphosites occurred in CDK consensus motifs, and 72% located to intrinsically disordered regions. Dynamically phosphorylated proteins, and documented substrates of cell cycle kinases, are significantly more disordered than phosphoproteins in general. Furthermore, 30-50% are components of membraneless organelles. Our results suggest that phosphorylation of intrinsically disordered proteins by cell cycle kinases, particularly CDKs, allows switch-like mitotic cellular reorganisation.

cell biology↗

bioRxiv2020

Multistep mechanism of DNA replication-coupled G-quadruplex resolution

G-quadruplex (or G4) structures are non-canonical DNA structures that form in guanine-rich sequences and threaten genome stability when not properly resolved. G4 unwinding occurs during S phase via an unknown mechanism. Using Xenopus egg extracts, we define a three-step G4 unwinding mechanism that is coupled to DNA replication. First, the replicative helicase (CMG) stalls at a leading strand G4 structure. Second, the DHX36 helicase mediates the bypass of the CMG past the intact G4 structure, which allows approach of the leading strand to the G4. Third, G4 structure unwinding by the FANCJ helicase enables the DNA polymerase to synthesize past the G4 motif. A G4 on the lagging strand template does not stall CMG, but still requires DNA replication for unwinding. DHX36 and FANCJ have partially redundant roles, conferring robustness to this pathway. Our data reveal a novel genome maintenance pathway that promotes faithful G4 replication thereby avoiding genome instability.

biochemistry↗

bioRxiv2018

HSF2BP Negatively Regulates Homologous Recombination in DNA Interstrand Crosslink Repair in Human Cells by Direct Interaction With BRCA2

The tumor suppressor BRCA2 is essential for homologous recombination, replication fork stability and DNA interstrand crosslink (ICL) repair in vertebrates. We show that a functionally uncharacterized protein, HSF2BP, is involved in a novel, direct and highly evolutionarily conserved interaction with BRCA2. Although HSF2BP was previously described as testis-specific, we find it is expressed in mouse ES cells, in human cancer cell lines, and in tumor samples. Elevated levels of HSF2BP sensitize human cells to ICL-inducing agents (mitomycin C and cisplatin) and PARP inhibitors, resulting in a phenotype characteristic of cells from Fanconi anemia (FA) patients. We biochemically recapitulate the suppression of ICL repair and establish that excess HSF2BP specifically compromises homologous recombination by preventing BRCA2 and RAD51 loading at the ICL. As increased ectopic expression of HSF2BP occurs naturally, we suggest that it can be considered as a causative agent in FA and a source of cancer-promoting genomic instability.

molecular biology↗