Biology subjects

Pillaire, M.-J.

Publications and source records attributed to Pillaire, M.-J..

4 recordsLinked to original sources

bioRxiv2024

Identification of the main barriers to Ku accumulation in chromatin

Repair of DNA double strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Given its high affinity for ends, multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to [~]1-2 molecules per DNA end. The mechanisms enforcing this limit are currently unknown. Here we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process which actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism which operates in S-phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together our data shed light on the multiple layers of coordinated mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions. HighlightsO_LIDNA-PKcs structurally blocks Ku sliding into chromatin in human & Xenopus C_LIO_LIA neddylation/FBXL12-dependent mechanism limits Ku accumulation on chromatin C_LIO_LIIn S-phase, ATM/CtIP overcomes Ku accumulation C_LIO_LIIn absence of DNA-PKcs, transcription at the DNA end vicinity is inhibited C_LI eTOC blurbThe DNA end binding protein Ku can slide onto naked DNA but this is limited in cells. Using human cells and Xenopus egg extracts, DNA-PKcs is identified as the main structural barrier to Ku entry into chromatin, along with two active mechanisms which limit Ku accumulation in absence of DNA-PKcs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/574002v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@4ebc45org.highwire.dtl.DTLVardef@12a7805org.highwire.dtl.DTLVardef@12db0a8org.highwire.dtl.DTLVardef@a925cc_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

Cytidine deaminase protects pancreatic cancer cells from replicative stress and drives resistance to DNA-targeting drugs

Chronic DNA replication stress and genome instability are two hallmarks of cancer that fuel oncogenesis and tumor diversity. Therapeutic approaches aimed to leverage tumor-specific replication stress to intolerable levels or to expose vulnerabilities for synthetic lethality purposes have recently gained momentum, especially for pancreatic cancer, a disease with no cure. However, the current knowledge regarding the molecular mechanisms involved in the replication stress response in pancreatic tumors is limited. Cytidine deaminase (CDA) is involved in the pyrimidine salvage pathway for DNA and RNA synthesis. Loss of CDA induces genomic instability in Bloom Syndrome, and CDA protects tumor cells from chemotherapy with pyrimidine analogs. Here, we show that CDA is overexpressed in genetically unstable pancreatic tumors, associates with a DNA replication signature, and is instrumental for experimental tumor growth. In cancer cells, CDA promotes DNA replication, increases replication fork speed, and controls replication stress and genomic stability levels. CDA expression is predictive of DNA-damaging drug efficacy and targeting CDA relieves resistance to chemotherapy in patients models, both in vitro and in vivo. Our findings shed new light on the mechanisms by which pancreatic cancer cells control replication stress, and highlight targeting of CDA as a potential therapeutic strategy to defeat tumor resistance to treatment.

cancer biology↗

bioRxiv2021

A catalytic-independent function of human DNA polymerase Kappa controls the pool of the Checkpoint Kinase 1

DNA polymerase kappa (Pol {kappa}) has been well documented thus far for its specialized DNA synthesis activity during translesion replication, progression of replication forks through regions difficult to replicate and replication checkpoint at stalled forks. Here we unveiled an unexpected role for Pol {kappa} in controlling the stability and abundance of Chk1, the major mediator of the replication checkpoint. We found that loss of Pol {kappa} decreased the Chk1 protein level in the nucleus of four human cell lines. Pol {kappa} and not the other Y-family polymerase members is required to maintain the Chk1 protein pool all along the cell cycle. We showed that Pol {kappa} depletion affected the protein stability of Chk1 and protected it from proteasome degradation and the replication recovery defects observed in Pol {kappa}-depleted cells could be overcome by the re-expression of Chk1. Importantly, this new function of Pol {kappa} does not require its catalytic activity, revealing that in addition to its known roles in the replication process, Pol {kappa} can contribute to the maintenance of genome stability independently of its DNA synthesis activity.

cell biology↗

bioRxiv2019

KDM5 histone-demethylases contribute to replication stress response and tolerance.

KDM5A and KDM5B histone-demethylases are overexpressed in many cancers and have been involved in drug tolerance. Here, we describe that KDM5A, together with KDM5B, contribute to replication stress (RS) response and tolerance. First, they positively regulate RRM2, the regulatory subunit of Ribonucleotide Reductase. Second, they are required for optimal activation of Chk1, a major player of the intra-S phase checkpoint that protects cells from RS. This role in Chk1 activation is probably direct since KDM5A is enriched at ongoing replication forks and associates with both PCNA and Chk1. Because RRM2 is a major determinant of replication stress tolerance, we developed cells resistant to HU, and show that KDM5A/B proteins are required for both RRM2 overexpression and tolerance to HU, in a manner that is independent of their demethylase activity. Altogether, our results indicate that KDM5A/B are major players of RS management. They also show that drugs targeting the enzymatic activity of KDM5 proteins may not affect all cancer-related consequences of KDM5A/B overexpression.

cancer biology↗