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The KU70-SAP domain has an overlapping function with DNA-PKcs in limiting the lateral movement of KU along DNA

ABSTRACT: The non-homologous end-joining (NHEJ) pathway is critical for DNA double-strand break repair and is essential for lymphocyte development and maturation. The Ku70/Ku80 heterodimer (KU) binds to DNA ends, initiating NHEJ and recruiting additional factors, including DNA-dependent protein kinase catalytic subunit (DNA-PKcs) that caps the ends and pushes KU inward. The C-terminus of Ku70 in higher eukaryotes includes a flexible linker and a SAP domain, whose physiological role remains poorly understood. To investigate this, we generated a mouse model with knock-in deletion of the SAP domain (Ku70DSAP/DSAP). Ku70DSAP supports KU stability and its recruitment to DNA damage sites in vivo. In contrast to the growth retardation and immunodeficiency seen in Ku70-/- mice, Ku70DSAP/DSAP mice show no defects in lymphocyte development and maturation. Structural modeling of KU on long dsDNA, but not dsRNA suggests that the SAP domain can bind to an adjacent major groove, where it can limit KU’s rotation and lateral movement along the dsDNA. Consistent with this model, in the absence of DNA-PKcs that caps the ends, Ku70DSAP fails to support stable DNA damage-induced KU foci. In DNA-PKcs-/- mice, Ku70DSAP abrogates the leaky T cell development and reduces both the qualitative and quantitative aspects of residual V(D)J recombination. Moreover, purified Ku70DSAP also has reduced affinity for DNA ends and dissociates more readily. These findings revealed a physiological role of the SAP domain in NHEJ by restricting KU rotation and lateral movement on DNA that is largely masked by DNA-PKcs.

ORGANISM(S): Mus musculus

PROVIDER: GSE276069 | GEO | 2024/12/31

REPOSITORIES: GEO

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The KU70-SAP domain has an overlapping function with DNA-PKcs in limiting the lateral movement of KU along DNA

Project description:The KU70-SAP domain has an overlapping function with DNA-PKcs in limiting the lateral movement of KU along DNA

| PRJNA1154631 | ENA

PAXX is an accessory c-NHEJ factor that associates with Ku70 and has overlapping functions with XLF

Project description:In mammalian cells, classical non-homologous end joining (c-NHEJ) is critical for DNA double-strand break repair induced by ionizing radiation and during V(D)J recombination in developing B and T lymphocytes. Recently, PAXX was identified as a new c-NHEJ core component. We report here, that PAXX-deficient cells exhibit a cellular phenotype uncharacteristic of a deficiency in c-NHEJ core components. PAXX-deficient cells display normal sensitivity to radiomimetic drugs, are proficient in transient V(D)J recombination assays, and do not shift towards higher micro-homology usage in plasmid repair assays. Although PAXX-deficient cells lack c-NHEJ phenotypes, PAXX forms a ternary complex with Ku bound to DNA. Formation of this complex involves an interaction with Ku70 and requires a bare DNA extension for stability. Moreover, the relatively weak Ku-dependent stimulation of LIG4/XRCC4 activity by PAXX is unmasked by XLF ablation. Thus, PAXX plays an accessory role during c-NHEJ that is largely overlapping with XLF’s function.

2016-10-06 | PXD004659 | Pride

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