Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase

M. M. Purugganan, S. Shah, J. F. Kearney, D. B. Roth

Research output: Contribution to journalArticle

Abstract

V(D)J recombination generates a remarkably diverse repertoire of antigen receptors through the rearrangement of germline DNA. Terminal deoxynucleotidyl transferase (TdT), a polymerase that adds random nucleotides (N regions) to recombination junctions, is a key enzyme contributing to this diversity. The current model is that TdT adds N regions during V(D)J recombination by random collision with the DNA ends, without a dependence on other cellular factors. We previously demonstrated, however, that V(D)J junctions from Ku80-deficient mice unexpectedly lack N regions, although the mechanism responsible for this effect remains undefined in the mouse system. One possibility is that junctions are formed in these mice during a stage in development when TdT is not expressed. Alternatively, Ku80 may be required for the expression, nuclear localization or enzymatic activity of TdT. Here we show that V(D)J junctions isolated from Ku80-deficient fibroblasts are devoid of N regions, as were junctions in Ku80-deficient mice. In these cells TdT protein is abundant at the time of recombination, localizes properly to the nucleus and is enzymatically active. Based on these data, we propose that TdT does not add to recombination junctions through random collision but is actively recruited to the V(D)J recombinase complex by Ku80.
Original languageUndefined
Pages (from-to)1638-46
JournalNucleic Acids Research
Volume29
Issue number7
StatePublished - 2001

Keywords

  • Animals *Antigens, Nuclear CHO Cells Cell Nucleus/enzymology Cricetinae *DNA Helicases DNA Nucleotidylexotransferase/genetics/*metabolism DNA-Binding Proteins/genetics/*metabolism Fibroblasts/cytology/metabolism Gene Rearrangement Genes, Immunoglobulin Homeodomain Proteins/genetics/metabolism Immunoglobulin Joining Region/*genetics/metabolism Immunoglobulin Variable Region/*genetics/metabolism Nuclear Proteins/genetics/*metabolism Nucleotides/*metabolism Plasmids/genetics Transfection

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Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase. / Purugganan, M. M.; Shah, S.; Kearney, J. F.; Roth, D. B.

In: Nucleic Acids Research, Vol. 29, No. 7, 2001, p. 1638-46.

Research output: Contribution to journalArticle

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abstract = "V(D)J recombination generates a remarkably diverse repertoire of antigen receptors through the rearrangement of germline DNA. Terminal deoxynucleotidyl transferase (TdT), a polymerase that adds random nucleotides (N regions) to recombination junctions, is a key enzyme contributing to this diversity. The current model is that TdT adds N regions during V(D)J recombination by random collision with the DNA ends, without a dependence on other cellular factors. We previously demonstrated, however, that V(D)J junctions from Ku80-deficient mice unexpectedly lack N regions, although the mechanism responsible for this effect remains undefined in the mouse system. One possibility is that junctions are formed in these mice during a stage in development when TdT is not expressed. Alternatively, Ku80 may be required for the expression, nuclear localization or enzymatic activity of TdT. Here we show that V(D)J junctions isolated from Ku80-deficient fibroblasts are devoid of N regions, as were junctions in Ku80-deficient mice. In these cells TdT protein is abundant at the time of recombination, localizes properly to the nucleus and is enzymatically active. Based on these data, we propose that TdT does not add to recombination junctions through random collision but is actively recruited to the V(D)J recombinase complex by Ku80.",
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T1 - Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase

AU - Purugganan, M. M.

AU - Shah, S.

AU - Kearney, J. F.

AU - Roth, D. B.

N1 - 1362-4962 Purugganan, M M Shah, S Kearney, J F Roth, D B AI36420/AI/NIAID NIH HHS/United States AI45794-01/AI/NIAID NIH HHS/United States R01 AI045794/AI/NIAID NIH HHS/United States Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. England Nucleic Acids Res. 2001 Apr 1;29(7):1638-46.

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N2 - V(D)J recombination generates a remarkably diverse repertoire of antigen receptors through the rearrangement of germline DNA. Terminal deoxynucleotidyl transferase (TdT), a polymerase that adds random nucleotides (N regions) to recombination junctions, is a key enzyme contributing to this diversity. The current model is that TdT adds N regions during V(D)J recombination by random collision with the DNA ends, without a dependence on other cellular factors. We previously demonstrated, however, that V(D)J junctions from Ku80-deficient mice unexpectedly lack N regions, although the mechanism responsible for this effect remains undefined in the mouse system. One possibility is that junctions are formed in these mice during a stage in development when TdT is not expressed. Alternatively, Ku80 may be required for the expression, nuclear localization or enzymatic activity of TdT. Here we show that V(D)J junctions isolated from Ku80-deficient fibroblasts are devoid of N regions, as were junctions in Ku80-deficient mice. In these cells TdT protein is abundant at the time of recombination, localizes properly to the nucleus and is enzymatically active. Based on these data, we propose that TdT does not add to recombination junctions through random collision but is actively recruited to the V(D)J recombinase complex by Ku80.

AB - V(D)J recombination generates a remarkably diverse repertoire of antigen receptors through the rearrangement of germline DNA. Terminal deoxynucleotidyl transferase (TdT), a polymerase that adds random nucleotides (N regions) to recombination junctions, is a key enzyme contributing to this diversity. The current model is that TdT adds N regions during V(D)J recombination by random collision with the DNA ends, without a dependence on other cellular factors. We previously demonstrated, however, that V(D)J junctions from Ku80-deficient mice unexpectedly lack N regions, although the mechanism responsible for this effect remains undefined in the mouse system. One possibility is that junctions are formed in these mice during a stage in development when TdT is not expressed. Alternatively, Ku80 may be required for the expression, nuclear localization or enzymatic activity of TdT. Here we show that V(D)J junctions isolated from Ku80-deficient fibroblasts are devoid of N regions, as were junctions in Ku80-deficient mice. In these cells TdT protein is abundant at the time of recombination, localizes properly to the nucleus and is enzymatically active. Based on these data, we propose that TdT does not add to recombination junctions through random collision but is actively recruited to the V(D)J recombinase complex by Ku80.

KW - Animals Antigens, Nuclear CHO Cells Cell Nucleus/enzymology Cricetinae DNA Helicases DNA Nucleotidylexotransferase/genetics/metabolism DNA-Binding Proteins/genetics/metabolism Fibroblasts/cytology/metabolism Gene Rearrangement Genes, Immunoglobulin Homeod

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