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. 1991 Aug;11(8):3972–3977. doi: 10.1128/mcb.11.8.3972

V(D)J recombination: evidence that a replicative mechanism is not required.

C L Hsieh 1, R P McCloskey 1, E Radany 1, M R Lieber 1
PMCID: PMC361195  PMID: 2072902

Abstract

We examined a series of extrachromosomal DNA substrates for V(D)J recombination under replicating and nonreplicating conditions. Complete and partial replications were examined by monitoring the loss of prokaryote-specific adenine methylation at 14 to 22 MboI-DpnI restriction sites (GATC) on the substrates. Some of these sites are within 2 bases of the signal sequence ends. We found that neither coding joint nor signal joint formation requires substrate replication. After ruling out replication as a substrate requirement, we determined whether replication had any effect on the efficiency of V(D)J recombination. Quantitation of V(D)J recombination efficiency on nonreplicating substrates requires some method of monitoring the entry of substrate molecules into the cells. We devised such a method by monitoring DNA repair of substrates into which we had substituted deoxyuridine for 10 to 20% of the thymidine nucleotides in the DNA. The substrates which enter the lymphoid cells were repaired efficiently in vivo by the eukaryotic uracil DNA repair system. Upon plasmid harvest, we distinguished repaired (entered) from unrepaired (not entered) plasmids by cleaving unrepaired molecules with uracil DNA glycoylase and Escherichia coli endonuclease IV in vitro. This method of monitoring DNA entry does not appear to underestimate or overestimate the amount of DNA entry. By using this method, we found no significant quantitative effect of DNA replication on V(D)J recombination efficiency.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alt F. W., Baltimore D. Joining of immunoglobulin heavy chain gene segments: implications from a chromosome with evidence of three D-JH fusions. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4118–4122. doi: 10.1073/pnas.79.13.4118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alt F. W., Blackwell T. K., DePinho R. A., Reth M. G., Yancopoulos G. D. Regulation of genome rearrangement events during lymphocyte differentiation. Immunol Rev. 1986 Feb;89:5–30. doi: 10.1111/j.1600-065x.1986.tb01470.x. [DOI] [PubMed] [Google Scholar]
  3. Early P., Huang H., Davis M., Calame K., Hood L. An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH. Cell. 1980 Apr;19(4):981–992. doi: 10.1016/0092-8674(80)90089-6. [DOI] [PubMed] [Google Scholar]
  4. Hesse J. E., Lieber M. R., Gellert M., Mizuuchi K. Extrachromosomal DNA substrates in pre-B cells undergo inversion or deletion at immunoglobulin V-(D)-J joining signals. Cell. 1987 Jun 19;49(6):775–783. doi: 10.1016/0092-8674(87)90615-5. [DOI] [PubMed] [Google Scholar]
  5. Hesse J. E., Lieber M. R., Mizuuchi K., Gellert M. V(D)J recombination: a functional definition of the joining signals. Genes Dev. 1989 Jul;3(7):1053–1061. doi: 10.1101/gad.3.7.1053. [DOI] [PubMed] [Google Scholar]
  6. Höchtl J., Müller C. R., Zachau H. G. Recombined flanks of the variable and joining segments of immunoglobulin genes. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1383–1387. doi: 10.1073/pnas.79.5.1383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Landau N. R., Schatz D. G., Rosa M., Baltimore D. Increased frequency of N-region insertion in a murine pre-B-cell line infected with a terminal deoxynucleotidyl transferase retroviral expression vector. Mol Cell Biol. 1987 Sep;7(9):3237–3243. doi: 10.1128/mcb.7.9.3237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Levin J. D., Johnson A. W., Demple B. Homogeneous Escherichia coli endonuclease IV. Characterization of an enzyme that recognizes oxidative damage in DNA. J Biol Chem. 1988 Jun 15;263(17):8066–8071. [PubMed] [Google Scholar]
  9. Lewis S., Gifford A., Baltimore D. DNA elements are asymmetrically joined during the site-specific recombination of kappa immunoglobulin genes. Science. 1985 May 10;228(4700):677–685. doi: 10.1126/science.3158075. [DOI] [PubMed] [Google Scholar]
  10. Lieber M. R., Hesse J. E., Lewis S., Bosma G. C., Rosenberg N., Mizuuchi K., Bosma M. J., Gellert M. The defect in murine severe combined immune deficiency: joining of signal sequences but not coding segments in V(D)J recombination. Cell. 1988 Oct 7;55(1):7–16. doi: 10.1016/0092-8674(88)90004-9. [DOI] [PubMed] [Google Scholar]
  11. Lieber M. R., Hesse J. E., Mizuuchi K., Gellert M. Developmental stage specificity of the lymphoid V(D)J recombination activity. Genes Dev. 1987 Oct;1(8):751–761. doi: 10.1101/gad.1.8.751. [DOI] [PubMed] [Google Scholar]
  12. Lieber M. R., Hesse J. E., Mizuuchi K., Gellert M. Lymphoid V(D)J recombination: nucleotide insertion at signal joints as well as coding joints. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8588–8592. doi: 10.1073/pnas.85.22.8588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Max E. E., Seidman J. G., Leder P. Sequences of five potential recombination sites encoded close to an immunoglobulin kappa constant region gene. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3450–3454. doi: 10.1073/pnas.76.7.3450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sakano H., Hüppi K., Heinrich G., Tonegawa S. Sequences at the somatic recombination sites of immunoglobulin light-chain genes. Nature. 1979 Jul 26;280(5720):288–294. doi: 10.1038/280288a0. [DOI] [PubMed] [Google Scholar]
  15. Sato K., Ito R., Baek K. H., Agarwal K. A specific DNA sequence controls termination of transcription in the gastrin gene. Mol Cell Biol. 1986 Apr;6(4):1032–1043. doi: 10.1128/mcb.6.4.1032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Schatz D. G., Oettinger M. A., Baltimore D. The V(D)J recombination activating gene, RAG-1. Cell. 1989 Dec 22;59(6):1035–1048. doi: 10.1016/0092-8674(89)90760-5. [DOI] [PubMed] [Google Scholar]
  17. Taylor A. F., Weiss B. Role of exonuclease III in the base excision repair of uracil-containing DNA. J Bacteriol. 1982 Jul;151(1):351–357. doi: 10.1128/jb.151.1.351-357.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]

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