Biology subjects

Frit, P.

Publications and source records attributed to Frit, P..

3 recordsLinked to original sources

bioRxiv2024

DNA polymerase Lambda is anchored within the NHEJ synaptic complex via Ku70/80

Non-homologous end joining (NHEJ) is the predominant pathway by which double-strand DNA breaks (DSBs) are repaired in mammals. To enable final break closure, various NHEJ end-processing factors respond to the chemistry of the damaged DNA ends. Amongst these factors is DNA polymerase lambda (Pol {lambda}), a member of the Pol X family. How members of the Pol X family engage with the NHEJ complex is unknown. Here, we present cryo-EM structures of Pol {lambda} in complex with the Ku70/80 DSB sensor whilst engaged with the DNA-PK holoenzyme in a long-range synaptic complex. These structures reveal a specific interaction site between Ku70/80 and the Pol {lambda} BRCT domain. The functionality of this interaction is assessed by generating point mutations on either side of the Pol {lambda} BRCT:Ku70/80 interface. Using these mutants in two orthogonal assays in cells (live protein recruitment at biphoton laser-damaged nuclear sites and transfection with an original gap-filling reporter plasmid) defines the molecular basis and essentiality of the BRCT domain for the recruitment and activity of the Pol {lambda} within the NHEJ complex. Ultimately, these data explain the role of this interaction in cell survival to DSBs. Finally, we propose a unified model for the interaction of the three Pol X family members bearing BRCT domains with the same site of Ku70/80.

biochemistry↗

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↗

bioRxiv2023

Ku-binding motifs in RAG2, XLF, PAXX and MRI support functional redundancy during V(D)J recombination

The interaction of several partners with Ku through Ku-binding motifs (KBMs) in their sequences governs their enrolment in NHEJ repair complexes. Here, we first established more specifically the function of KBMs in V(D)J recombination as the molecular basis of functional redundancy between XLF and the NHEJ proteins MRI and PAXX. Then, given the functional redundancy between RAG2 and XLF, we explored the hypothesis of a KBM-mediated interaction between RAG2 and Ku. Through sequence alignment and biophysical methods, we identified a KBM at the C-terminus of RAG2 (R2CT) that mediates its interaction with Ku both in vitro and in cellulo. Notably, we showed that R2CT/Ku interaction is independent of the RAG nuclease activity. Finally, we demonstrated that the respective KBMs of RAG2 and XLF support their functional redundancy for V(D)J recombination.

molecular biology↗