Lig4 modulates p53-p21 checkpoints and inhibits A-EJ during end-joining repair phase of IgH class switching (LYM6P.770)

Abstract

Abstract Mutations in the non-homologous end-joining DNA repair protein DNA Ligase IV (LIG4) causes a human syndrome of immunodeficiency and radiosensitivity. We previously described knock-in mice with a homozygous R278H mutation in Lig4 (Lig4R/R) that displayed severely leaky B lymphocyte development, resulting in near lack of mature B cells yet only a partial block in IgH class switch recombination (CSR). Here we show that diminished DNA ligase 4 activity, resulting in slower kinetics of DNA double strand break (DSB) repair and attenuation of p53-dependent DNA double strand break (DSB) response, to underlie the partial CSR block in the Lig4R/R activated B cells. Analysis of p53 levels in activated Lig4R/R B cells revealed increased proteosome mediated p53 decay and p21 activation, indicative of slower repair kinetics, effects mimicked in the human Lig4 R278H 180BR cell line. Concurrent p53 inactivation had little effect on the frequency of IgH breaks; but substantially slowed the cell cycle progression of activated Lig4R/Rp53-/- B cells, and failed to promote the development of B-lymphomas that recurrently developed in murine Lig4-/-p53-/- settings. Analysis Lig4R/R CSR junctions also revealed the predominance of NHEJ instead of NHEJ-independent A-EJ in their generation. Hence NHEJ maintains genomic stability by modulating the p53 DNA damage response and inhibiting A-EJ. These findings provide an explanation for the complex B-lymphoid phenotypes of the human Lig4 Syndrome.