Cell-cycle-regulated dna double-strand breaks in somatic hypermutation of immunoglobulin genes

ABSTRACT Targeted hypermutation of immunoglobulin variable region genes occurs in B cells during an immune response1, and gives rise to families of related mutant antibodies which are then

selected for their binding affinity to the immunizing antigen2. Somatic hypermutation predominantly generates point mutations, many of which occur at specific residues (hotspots)3. The

reaction has been linked to transcription and requires the presence of immunoglobulin enhancers4,5,6, but replacement of the variable gene by heterologous sequences, or the variable region

promoter by a heterologous promoter, does not interfere with the mutation process7,8. Here we show the existence of abundant DNA double-strand breaks (DSBs) in hypermutating sequences.

Generation of the DSBs is coupled to transcription, enhancer-dependent, and correlates with the appearance of nearby mutations. Furthermore, the DSBs are cell-cycle restricted, being found

almost exclusively in cells that have completed, or nearly completed, DNA replication. We propose a model for somatic hypermutation in which mutations are introduced into the DNA during

repair of DSBs by homologous recombination. The finding of DSBs during somatic hypermutation may help to explain the chromosomal translocations found in some B-cell tumours. Access through

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BEING VIEWED BY OTHERS SUPER-ENHANCER HYPERMUTATION ALTERS ONCOGENE EXPRESSION IN B CELL LYMPHOMA Article 06 July 2022 REGULATED SOMATIC HYPERMUTATION ENHANCES ANTIBODY AFFINITY MATURATION

Article Open access 19 March 2025 TRANSIENT SILENCING OF HYPERMUTATION PRESERVES B CELL AFFINITY DURING CLONAL BURSTING Article Open access 19 March 2025 REFERENCES * Rajewsky, K. Clonal

selection and learning in the antibody system. _Nature_ 381, 751–758 (1996). Article  ADS  CAS  Google Scholar  * Weigert, M., Cesari, I., Yonkovich, S. & Cohn, M. Variability in the

lambda light chain sequences of mouse antibody. _Nature_ 228, 1045–1047 (1970). Article  ADS  CAS  Google Scholar  * Neuberger, M. S. _ et al._ Monitoring and interpreting the intrinsic

features of somatic hypermutation. _Immunol. Rev._ 162, 107– 116 (1998). Article  CAS  Google Scholar  * Peters, A. & Storb, U. Somatic hypermutation of immunoglobulin genes is linked to

transcription initiation. _Immunity_ 4, 57–65 (1996). Article  CAS  Google Scholar  * Goyenechea, B. _ et al._ Cells strongly expressing Igκ transgenes show clonal recruitment of

hyper-mutation: a role for both MAR and the enhancers. _ EMBO J._ 16, 3987–3994 ( 1997). Article  CAS  Google Scholar  * Fukita, Y., Jacobs, H. & Rajewsky, K. Somatic hypermutation in

the heavy chain locus correlates with transcription. _Immunity_ 9, 105– 114 (1998). Article  CAS  Google Scholar  * Yelamos, J. _ et al._ Targeting of non-Ig sequences in place of the V

segment by somatic hypermutation. _Nature_ 376, 225– 229 (1995). Article  ADS  CAS  Google Scholar  * Tumas-Brundage, K. & Manser, T. The transcriptional promoter regulates hypermutation

of the antibody heavy chain locus. _ J. Exp. Med._ 185, 239–250 (1997). Article  CAS  Google Scholar  * Brenner, S. & Milstein, C. Origin of antibody variation. _ Nature_ 211, 242–243 (

1966). Article  ADS  CAS  Google Scholar  * Lo, A. K., Ching, A. K., Lim, P. L. & Chui, Y. L. Strand breaks in immunoglobulin gene hypermutation. _Ann. NY Acad. Sci._ 815, 432–435

(1997). Article  ADS  CAS  Google Scholar  * Sale, J. E. & Neuberger, M. S. TdT-accessible breaks are scattered over the immunoglobulin variable domain in a constitutively hypermutating

B cell line. _Immunity_ 9, 859– 869 (1998). Article  CAS  Google Scholar  * Schlissel, M., Constantinescu, A., Morrow, T., Baxter, M. & Peng, A. Double-strand signal sequence breaks in

V(D)J recombination are blunt, 5′-phosphorylated, RAG-dependent, and cell cycle regulated. _Genes Dev._ 7, 2520–2532 (1993). Article  CAS  Google Scholar  * Weiss, S. & Wu, G. E. Somatic

point mutations in unrearranged immunoglobulin gene segments encoding the variable region of lambda light chains. _EMBO J._ 6, 927– 932 (1987). Article  CAS  Google Scholar  * Vignali, D.

A., Carson, R. T., Chang, B., Mittler, R. S. & Strominger, J. L. The two membrane proximal domains of CD4 interact with the T cell receptor. _J. Exp. Med._ 183, 2097–2107 (1996). Article

  CAS  Google Scholar  * Jeggo, P. A. Studies on mammalian mutants defective in rejoining double-strand breaks in DNA. _Mutat. Res._ 239, 1– 16 (1990). Article  CAS  Google Scholar  *

Hendrickson, E. A. Insights from model systems: Cell-cycle regulation of mammalian DNA double-strand-break repair. _Am. J. Hum. Genet._ 61, 795– 800 (1997). Article  CAS  Google Scholar  *

Takata, M. _ et al._ Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in

vertebrate cells. _EMBO J._ 17, 5497–5508 (1998). Article  CAS  Google Scholar  * Strathern, J. N., Shafer, B. K. & McGill, C. B. DNA synthesis errors associated with double-strand-break

repair. _Genetics_ 140, 965– 972 (1995). CAS  PubMed  PubMed Central  Google Scholar  * Holbeck, S. L. & Strathern, J. N. A role for REV3 in mutagenesis during double-strand break

repair in _Saccharomyces cerevisiae_. _Genetics_ 147, 1017– 1024 (1997). CAS  PubMed  PubMed Central  Google Scholar  * Johnson, R. E., Washington, M. T., Haracska, L., Prakash, S. &

Prakash, L. Eukaryotic polymerases iota and zeta act sequentially to bypass DNA lesions. _Nature_ 406, 1015–1019 (2000). Article  ADS  CAS  Google Scholar  * Tissier, A. _ et al._

Misinsertion and bypass of thymine-thymine dimers by human DNA polymerase iota. _EMBO J._ 19, 5259– 5266 (2000). Article  CAS  Google Scholar  * Goossens, T., Klein, U. & Kuppers, R.

Frequent occurrence of deletions and duplications during somatic hypermutation: implications for oncogene translocations and heavy chain disease. _Proc. Natl Acad. Sci. USA_ 95, 2463–2468

(1998). Article  ADS  CAS  Google Scholar  * Wilson, P. C. _ et al._ Somatic hypermutation introduces insertions and deletions into immunoglobulin V genes. _J. Exp. Med._ 187, 59–70 (1998).

Article  CAS  Google Scholar  * Klein, U. _et al._ Somatic hypermutation in normal and transformed human B cells. _Immunol. Rev._ 162, 261– 280 (1998). Article  CAS  Google Scholar  *

Kenter, A. L. The liaison of isotype class switch and mismatch repair: an illegitimate affair. _J. Exp. Med._ 190, 307– 310 (1999). Article  CAS  Google Scholar  * Fugmann, S. D., Lee, A.

I., Shockett, P. E., Villey, I. J. & Schatz, D. G. The RAG proteins and V(D)J recombination: complexes, ends, and transposition. _Annu. Rev. Immunol._ 18, 495–527 (2000). Article  CAS 

Google Scholar  * Petrie, H. T., Livak, F., Burtrum, D. & Mazel, S. T cell receptor gene recombination patterns and mechanisms: cell death, rescue, and T cell production. _J. Exp. Med._

182, 121– 127 (1995). Article  CAS  Google Scholar  * Rada, C., Ehrenstein, M. R., Neuberger, M. S. & Milstein, C. Hot spot focusing of somatic hypermutation in MSH2-deficient mice

suggests two stages of mutational targeting. _Immunity_ 9, 135–141 (1998). Article  CAS  Google Scholar  * McGill, C. B., Holbeck, S. L. & Strathern, J. N. The chromosome bias of

misincorporations during double-strand break repair is not altered in mismatch repair-defective strains of _Saccharomyces cerevisiae_. _Genetics_ 148, 1525– 1533 (1998). CAS  PubMed  PubMed

Central  Google Scholar  * Paques, F. & Haber, J. E. Multiple pathways of recombination induced by double-strand breaks in _Saccharomyces cerevisiae_. _ Microbiol. Mol. Biol. Rev._ 63,

349– 404 (1999). CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We thank T. Taylor and G. Tokmoulina for help with cell sorting; E. Hilton for special

assistance with DNA sequencing; C. Arthur for her role in the creation of transgenic mice; and S. Fugmann and M. Diaz for many helpful discussions. We are very grateful to the following

people for helpful comments on the manuscript: M. Diaz, S. Fugmann, J. Haber, D. Hesslein, M. Jasin, M. Nussenzweig and I. Villey. Oligonucleotide synthesis and DNA sequencing were performed

by the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University. F.N.P. was supported by a postdoctoral fellowship from the Arthritis Foundation and D.G.S. is an associate

investigator of the Howard Hughes Medical Institute. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of

Medicine, Box 208011, 310 Cedar Street , New Haven, 06520-8011, Connecticut, USA F. Nina Papavasiliou & David G. Schatz Authors * F. Nina Papavasiliou View author publications You can

also search for this author inPubMed Google Scholar * David G. Schatz View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence

to David G. Schatz. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TABLES 1 — 3 (DOC 26 KB) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Papavasiliou, F.,

Schatz, D. Cell-cycle-regulated DNA double-strand breaks in somatic hypermutation of immunoglobulin genes. _Nature_ 408, 216–221 (2000). https://doi.org/10.1038/35041599 Download citation *

Received: 31 July 2000 * Accepted: 02 October 2000 * Issue Date: 09 November 2000 * DOI: https://doi.org/10.1038/35041599 SHARE THIS ARTICLE Anyone you share the following link with will be

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