Biology subjects

Maric, C.

Publications and source records attributed to Maric, C..

5 recordsLinked to original sources

bioRxiv2025

PARP inhibitors affect the replisome and replication fork progression during a single S phase.

Poly(ADP-ribosyl)ation (PARylation) is a protein modification mostly synthesised and degraded by PARP1/2 and PARG enzymes, respectively. PARylation is involved in many covalent and non-covalent protein-protein interactions in the nucleus, making it a powerful form of molecular signaling in DNA metabolism. PARP inhibitors (PARPi) have shown efficacy in the treatment of Homologous Recombination (HR)-deficient cancers, yet the full range of molecular mechanisms underlying the activity of these drugs is not fully understood. Here, we decipher the direct consequences of PARPi-induced loss of PARylation on DNA replication. First, PARPi treatment during a single S phase induces replicative stress, delays S phase progression and causes genome-wide replication timing changes of disease-associated regions. These DNA replication alterations appear to be caused by an accumulation of SSBs in the DNA replicative template when PARylation is inhibited, which then seems to lead to one-ended DSBs and fork collapse during S phase. Second, PARPi deplete FANCD2-I, BRCA, monoubiquitinated PCNA and RAD18 proteins from the replisomes. The two PARPi tested then appear to modulate the choice of fork restart mechanisms and influence replisome dynamics in different ways. Taken together, our study highlights the common and unique primary effects of two PARPi on unperturbed DNA replication in human cells. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/639283v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@298f8dorg.highwire.dtl.DTLVardef@8f8cbcorg.highwire.dtl.DTLVardef@60952forg.highwire.dtl.DTLVardef@e2700a_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

DBF4, not DRF1, is the crucial regulator of CDC7 kinase at replication forks

In eukaryotes, CDC7 kinase is crucial for DNA replication initiation and has been involved in fork processing and replication stress response. Human CDC7 requires the binding of either one of two regulatory subunits, DBF4 and DRF1, for its activity. However, it is unclear whether the two regulatory subunits target CDC7 to a specific set of substrates, thus having different biological functions, or if they act redundantly. Using genome editing technology, we generated an isogenic set of cell lines deficient in either one of the two CDC7-activating subunits: these cells are viable but present signs of genomic instability, indicating that both DBF4 and DRF1 can independently support CDC7 for bulk DNA replication. Nonetheless, DBF4-deficient cells show altered replication efficiency, including partial deficiency in MCM helicase phosphorylation and alterations in the replication timing of discrete genomic regions. Notably, we find that CDC7 function at replication forks is entirely dependent on DBF4 and not DRF1. Thus, DBF4 is the primary regulator of CDC7 activity, likely mediating most of its functions in unperturbed DNA replication and during replication fork processing upon replication interference.

biochemistry↗

bioRxiv2022

Palbociclib interferes with replication origin firing in a pRb independent manner

Over the last decade, CDK4/6 inhibitors (palbociclib, ribociclib and abemaciclib) have emerged as promising anticancer drugs. Numerous studies have demonstrated that CDK4/6 inhibitors efficiently block the pRb-E2F pathway and induce cell cycle arrest in pRb-proficient cells. Based on these studies, the inhibitors have been approved by the FDA for treatment of advanced hormonal receptor (HR) positive breast cancers in combination with hormonal therapy. However, some evidence has recently shown unexpected effects of the inhibitors, promoting needs to understand more about the mechanism of inhibitors beyond pRb. Our study demonstrates here how palbociclib impairs the origin firing in the DNA replication process in pRb-deficient cell lines. Strikingly, despite the absence of pRb, cells treated with palbociclib synthesize less DNA without any induced cell cycle arrest. Furthermore, palbociclib treatment disturbs the temporal program of DNA replication and reduces the density of replication forks. Cells treated with palbociclib show a defect in the loading of proteins of the Pre-initiation complex (Pre-IC) on chromatin, indicating a reduced initiation of DNA replication. Our findings highlight hidden effects of palbociclib on the dynamics of DNA replication and on its cytotoxic consequences on cell viability in the absence of pRb. This study provides a potential therapeutic application of palbociclib to target genomic instability towards pRb deficient patients. Significance StatementPalbociclib is a promising anticancer drug for pRb-proficient cell, particularly for hormonal receptor positive breast cancer, that induces the cell cycle arrest. But what about pRb deficient cell lines ? Our results show that Palbociclib disturb the DNA replication process inducing a replicative stress, an increase of DNA damages and leading to a significant decrease in cell viability. Palbociclib impairs the DNA synthesis reducing the number of active origins with the decrease of availability of the pre-initiation complexes. We believe that the demonstration of this effect of palbociclib on pRb-deficient cells may be a new therapeutic entry point in combination with other treatments for these types of cancer. Replicative stress can be one of weaknesses of pRb defficient cancer cells.

cancer biology↗

bioRxiv2020

Low replication stress leads to specific replication timing advances associated to chromatin remodelling in cancer cells

DNA replication is well orchestrated in mammalian cells through a tight regulation of the temporal order of replication origin activation, named the replication timing, a robust and conserved process in each cell type. Upon low replication stress, the slowing of replication forks induces delayed replication of fragile regions leading to genetic instability. The impact of low replication stress on the replication timing in different cellular backgrounds has not been explored yet. Here we analysed the whole genome replication timing in a panel of 6 human cell lines under low replication stress. We first demonstrated that cancer cells were more impacted than non-tumour cells. Strikingly, we unveiled an enrichment of specific replication domains undergoing a switch from late to early replication in some cancer cells. We found that advances in replication timing correlate with heterochromatin regions poorly sensitive to DNA damage signalling while being subject to an increase of chromatin accessibility. Finally, our data indicate that, following release from replication stress conditions, replication timing advances can be inherited by the next cellular generation, suggesting a new mechanism by which cancer cells would adapt to cellular or environmental stress.

cancer biology↗

bioRxiv2019

New, easy, quick and efficient DNA replication timing analysis by high-throughput approaches.

DNA replication must be faithful and follow a well-defined spatio-temporal program closely linked to transcriptional activity, epigenomic marks, intra-nuclear structures, mutation rate and cell fate determination. Among the readouts of the DNA replication spatio-temporal program, replication timing (RT) analyses require complex, precise and time-consuming experimental procedures, and the study of large-size computer files. We improved the RT protocol to speed it up and increase its quality and reproducibility. Also, we partly automated the RT protocol and developed a user-friendly software: the START-R suite (Simple Tool for the Analysis of the Replication Timing based on R). START-R suite is an open source web application using an R script and an HTML interface to analyze DNA replication timing in a given cell line with microarray or deep-sequencing results. This novel approach can be used by every biologist without requiring specific knowledge in bioinformatics. It also reduces the time required for generating and analyzing simultaneously data from several samples. START-R suite detects constant timing regions (CTR) but also, and this is a novelty, it identifies temporal transition regions (TTR) and detects significant differences between two experimental conditions. The informatic global analysis requires less than 10 minutes.

genomics↗