Lymphocyte development requires ordered assembly and subsequent modifications of the antigen receptor genes through V(D)J recombination and Immunoglobulin class switch recombination (CSR), respectively. While the programmed DNA cleavage events are initiated by lymphocytespecific factors, the resulting DNA double-strand break (DSB) intermediates activate the ATM kinase-mediated DNA damage response (DDR) and rely on the ubiquitously expressed classical non-homologous end-joining (cNHEJ) pathway including the DNA-dependent protein kinase (DNA-PK), and, in the case of CSR, also the alternative end-joining (Alt-EJ) pathway, for repair. Correspondingly, patients and animal models with cNHEJ or DDR defects develop distinct types of immunodeficiency reflecting their specific DNA repair deficiency. The unique end-structure, sequence context, and cell cycle regulation of V(D)J recombination and CSR also provide a valuable platform to study the mechanisms of, and the interplay between, cNHEJ and DDR. Here, we compare and contrast the genetic consequences of DNA repair defects in V(D)J recombination and CSR with a focus on the newly discovered cNHEJ factors and the kinase-dependent structural roles of ATM and DNA-PK in animal models. Throughout, we try to highlight the pending questions and emerging differences that will extend our understanding of cNHEJ and DDR in the context of primary immunodeficiency and lymphoid malignancies.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a classical nonhomologous end-joining (cNHEJ) factor. Loss of DNA-PKcs diminished mature B cell class switch recombination (CSR) to other isotypes, but not IgG1. Here, we show that expression of the kinase-dead DNA-PKcs ( ) severely compromises CSR to IgG1. High-throughput sequencing analyses of CSR junctions reveal frequent accumulation of nonproductive interchromosomal translocations, inversions, and extensive end resection in , but not , B cells. Meanwhile, the residual joints from cells and the efficient Sµ-Sγ1 junctions from B cells both display similar preferences for small (2-6 nt) microhomologies (MH). In cells, Sµ-Sγ1 joints are more resistant to inversions and extensive resection than Sµ-Sε and Sµ-Sµ joints, providing a mechanism for the isotype-specific CSR defects. Together, our findings identify a kinase-dependent role of DNA-PKcs in suppressing MH-mediated end joining and a structural role of DNA-PKcs protein in the orientation of CSR.

analyzed the DNA-PKcs PQR , DNA-PKcs SD mouse and cells for lymphocyte development and function. W.J. generated the DNA-PKcs SD and DNA-PKcs PQR mice. X.S.W., J.L.C., Z.S., and B.J.L. established the HTGTS assay and performed the high-throughput genomic translocation sequence analyses of the class switch recombination junctions for this study. X.L. analyzed the V(D)J recombination junction in the DNA-PKcs SD and DNA-PKcs PQR mice.The high-throughput genomic translocation sequence data reported in this paper have been deposited in the Gene Expression Omnibus database (https://www.ncbi.nlm.nih. gov/geo/) under accession numbers GSE117628 and GSE129895.