CtIP-mediated DNA resection is dispensable for IgH class switch recombination by alternative end-joining

Significance Ig CSR occurs through the nonhomologous end-joining pathway. In NHEJ-deficient cells, substantial CSR can be achieved through the Alt-EJ pathway. High-throughput analyses of IgH CSR junctions in NHEJ-deficient cells show that in contrast to endonuclease-generated breaks, Alt-EJ-mediated CSR can occur without CtIP-mediated DNA resection. The first temporal analyses of CSR junctions in NHEJ-proficient cells further showed that early Sμ internal deletion junctions are enriched for MH. Taken together, the results identified repetitive core IgH switch regions as favorable substrates for MH-mediated recombination independent of resection. To generate antibodies with different effector functions, B cells undergo Immunoglobulin Heavy Chain (IgH) class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical nonhomologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable ∼25% of CSR can be achieved by the alternative end-joining (Alt-EJ) pathway that preferentially uses microhomology (MH) at the junctions. While A-EJ-mediated repair of endonuclease-generated breaks requires DNA end resection, we show that CtIP-mediated DNA end resection is dispensable for A-EJ-mediated CSR using cNHEJ-deficient B cells. High-throughput sequencing analyses revealed that loss of ATM/ATR phosphorylation of CtIP at T855 or ATM kinase inhibition suppresses resection without altering the MH pattern of the A-EJ-mediated switch junctions. Moreover, we found that ATM kinase promotes Alt-EJ-mediated CSR by suppressing interchromosomal translocations independent of end resection. Finally, temporal analyses reveal that MHs are enriched in early internal deletions even in cNHEJ-proficient B cells. Thus, we propose that repetitive IgH switch regions represent favored substrates for MH-mediated end-joining contributing to the robustness and resection independence of A-EJ-mediated CSR.

[1]  F. Alt,et al.  Fundamental Roles of Chromatin Loop Extrusion in Antibody Class Switching , 2019, Nature.

[2]  M. Lieber,et al.  Current insights into the mechanism of mammalian immunoglobulin class switch recombination , 2019, Critical reviews in biochemistry and molecular biology.

[3]  S. Zha,et al.  CtIP is essential for early B cell proliferation and development in mice , 2019, The Journal of experimental medicine.

[4]  L. Symington,et al.  Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection , 2018, Proceedings of the National Academy of Sciences.

[5]  F. Alt,et al.  Kinase-dependent structural role of DNA-PKcs during immunoglobulin class switch recombination , 2018, Proceedings of the National Academy of Sciences.

[6]  F. Alt,et al.  DNA double-strand break response factors influence end-joining features of IgH class switch and general translocation junctions , 2018, Proceedings of the National Academy of Sciences.

[7]  Richard L. Frock,et al.  Detecting DNA double-stranded breaks in mammalian genomes by linear amplification–mediated high-throughput genome-wide translocation sequencing , 2016, Nature Protocols.

[8]  T. Ludwig,et al.  The DNA resection protein CtIP promotes mammary tumorigenesis , 2016, Oncotarget.

[9]  K. Schlacher,et al.  A Selective Small Molecule DNA2 Inhibitor for Sensitization of Human Cancer Cells to Chemotherapy , 2016, EBioMedicine.

[10]  Kefei Yu,et al.  Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination , 2016, Proceedings of the National Academy of Sciences.

[11]  L. Symington,et al.  Microhomology-Mediated End Joining: A Back-up Survival Mechanism or Dedicated Pathway? , 2015, Trends in biochemical sciences.

[12]  Michel C. Nussenzweig,et al.  Orientation-Specific Joining of AID-initiated DNA Breaks Promotes Antibody Class Switching , 2015, Nature.

[13]  F. Alt,et al.  Related Mechanisms of Antibody Somatic Hypermutation and Class Switch Recombination , 2015, Microbiology spectrum.

[14]  Jeremy M. Stark,et al.  DNA Damage Response Factors from Diverse Pathways, Including DNA Crosslink Repair, Mediate Alternative End Joining , 2015, PLoS genetics.

[15]  S. Jackson,et al.  CtIP tetramer assembly is required for DNA-end resection and repair , 2014, Nature Structural &Molecular Biology.

[16]  D. Wesemann,et al.  Molecular Mechanisms of IgE Class Switch Recombination. , 2015, Current topics in microbiology and immunology.

[17]  Hailong Wang,et al.  Catalytic and noncatalytic roles of the CtIP endonuclease in double-strand break end resection. , 2014, Molecular cell.

[18]  T. Ludwig,et al.  CtIP-mediated resection is essential for viability and can operate independently of BRCA1 , 2014, The Journal of experimental medicine.

[19]  J. Chaudhuri,et al.  Regulation of immunoglobulin class-switch recombination: choreography of noncoding transcription, targeted DNA deamination, and long-range DNA repair. , 2014, Advances in immunology.

[20]  Mian Zhou,et al.  Mammalian DNA2 helicase/nuclease cleaves G‐quadruplex DNA and is required for telomere integrity , 2013, The EMBO journal.

[21]  B. Chait,et al.  Activation of DSB processing requires phosphorylation of CtIP by ATR. , 2013, Molecular cell.

[22]  Linda Z. Shi,et al.  The Interaction of CtIP and Nbs1 Connects CDK and ATM to Regulate HR–Mediated Double-Strand Break Repair , 2013, PLoS genetics.

[23]  T. Ludwig,et al.  Mechanism of DNA resection during intrachromosomal recombination and immunoglobulin class switching , 2012, The Journal of experimental medicine.

[24]  Ira M. Hall,et al.  YAHA: fast and flexible long-read alignment with optimal breakpoint detection , 2012, Bioinform..

[25]  F. Alt,et al.  Robust chromosomal DNA repair via alternative end-joining in the absence of X-ray repair cross-complementing protein 1 (XRCC1) , 2012, Proceedings of the National Academy of Sciences.

[26]  D. Ferguson,et al.  Mre11 regulates CtIP–dependent double strand break repair by interaction with CDK2 , 2011, Nature Structural &Molecular Biology.

[27]  F. Alt,et al.  Immature B cells preferentially switch to IgE with increased direct Sμ to Sε recombination , 2011, The Journal of experimental medicine.

[28]  L. Symington,et al.  Double-strand break end resection and repair pathway choice. , 2011, Annual review of genetics.

[29]  Jayanta Chaudhuri,et al.  CtIP promotes microhomology-mediated alternative end-joining during class switch recombination , 2010, Nature Structural &Molecular Biology.

[30]  R. Greenberg,et al.  ATM-Dependent Chromatin Changes Silence Transcription In cis to DNA Double-Strand Breaks , 2010, Cell.

[31]  M. Lieber,et al.  The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. , 2010, Annual review of biochemistry.

[32]  F. Alt,et al.  Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4 , 2010, The Journal of experimental medicine.

[33]  Katherine S. Yang-Iott,et al.  Formation of dynamic gamma-H2AX domains along broken DNA strands is distinctly regulated by ATM and MDC1 and dependent upon H2AX densities in chromatin. , 2009, Molecular cell.

[34]  Kefei Yu,et al.  Altered kinetics of nonhomologous end joining and class switch recombination in ligase IV–deficient B cells , 2008, The Journal of Experimental Medicine.

[35]  M. McVey,et al.  MMEJ repair of double-strand breaks (director's cut): deleted sequences and alternative endings. , 2008, Trends in genetics : TIG.

[36]  Jeremy M. Stark,et al.  Alternative-NHEJ Is a Mechanistically Distinct Pathway of Mammalian Chromosome Break Repair , 2008, PLoS genetics.

[37]  Jiri Bartek,et al.  Human CtIP promotes DNA end resection , 2007, Nature.

[38]  Michael M. Murphy,et al.  IgH class switching and translocations use a robust non-classical end-joining pathway , 2007, Nature.

[39]  F. Alt,et al.  Evolution of the immunoglobulin heavy chain class switch recombination mechanism. , 2007, Advances in immunology.

[40]  F. Alt,et al.  Pathways that suppress programmed DNA breaks from progressing to chromosomal breaks and translocations. , 2006, DNA repair.

[41]  F. Liu,et al.  Inactivation of CtIP Leads to Early Embryonic Lethality Mediated by G1 Restraint and to Tumorigenesis by Haploid Insufficiency , 2005, Molecular and Cellular Biology.

[42]  K. Rajewsky,et al.  Interference with Immunoglobulin (Ig)α Immunoreceptor Tyrosine–Based Activation Motif (Itam) Phosphorylation Modulates or Blocks B Cell Development, Depending on the Availability of an Igβ Cytoplasmic Tail , 2001, The Journal of experimental medicine.

[43]  R. Baer,et al.  Nuclear Localization and Cell Cycle-specific Expression of CtIP, a Protein That Associates with the BRCA1 Tumor Suppressor* , 2000, The Journal of Biological Chemistry.

[44]  F. Alt,et al.  A Critical Role for DNA End-Joining Proteins in Both Lymphogenesis and Neurogenesis , 1998, Cell.

[45]  K. Rajewsky,et al.  Receptor editing in a transgenic mouse model: site, efficiency, and role in B cell tolerance and antibody diversification. , 1997, Immunity.