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Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4.

Boboila C, Yan C, Wesemann DR, Jankovic M, Wang JH, Manis J, Nussenzweig A, Nussenzweig M, Alt FW - J. Exp. Med. (2010)

Bottom Line: Ku70- or Ku80-deficient B cells have reduced, but still substantial, CSR.Ku-deficient or Ku- plus Lig4-deficient B cells are also biased toward MH-mediated CSR joins; but, in contrast to XRCC4- or Lig4-deficient B cells, generate substantial numbers of direct CSR joins.Our findings suggest that more than one form of A-EJ can function in CSR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The classical nonhomologous end-joining (C-NHEJ) DNA double-strand break (DSB) repair pathway employs the Ku70/80 complex (Ku) for DSB recognition and the XRCC4/DNA ligase 4 (Lig4) complex for ligation. During IgH class switch recombination (CSR) in B lymphocytes, switch (S) region DSBs are joined by C-NHEJ to form junctions either with short microhomologies (MHs; "MH-mediated" joins) or no homologies ("direct" joins). In the absence of XRCC4 or Lig4, substantial CSR occurs via "alternative" end-joining (A-EJ) that generates largely MH-mediated joins. Because upstream C-NHEJ components remain in XRCC4- or Lig4-deficient B cells, residual CSR might be catalyzed by C-NHEJ using a different ligase. To address this, we have assayed for CSR in B cells deficient for Ku70, Ku80, or both Ku70 and Lig4. Ku70- or Ku80-deficient B cells have reduced, but still substantial, CSR. Strikingly, B cells deficient for both Ku plus Lig4 undergo CSR similarly to Ku-deficient B cells, firmly demonstrating that an A-EJ pathway distinct from C-NHEJ can catalyze CSR end-joining. Ku-deficient or Ku- plus Lig4-deficient B cells are also biased toward MH-mediated CSR joins; but, in contrast to XRCC4- or Lig4-deficient B cells, generate substantial numbers of direct CSR joins. Our findings suggest that more than one form of A-EJ can function in CSR.

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The Ku-dependent A-EJ pathway uses a distinct mechanism for S region joining. (a) Classical NHEJ performs both direct (left) and MH-mediated (right) end-joining. Direct joining occurs when DNA ends are joined either immediately, or after processing (removal of overhangs or fill-in by polymerases). MH-mediated joining (right) is facilitated by base-pairing interactions between short stretches of nucleotides at or near the ends of the DSB. (b) MH length in Sμ-Sγ1 (left) and Sμ-Sε (right) junctions from Ku70−/−HL, Ku70−/−Lig4−/−HL, Lig4−/−HL and WT/HL B cells stimulated in culture with αCD40 plus IL-4. Ku70-, Ku70/Lig4-, and Lig4-deficient cells have more Sμ-Sγ1 (left) and Sμ-Sε (right) joins with longer MH than WT. Data shown are based on 86 or more independent sequences per genotype for Sμ-Sγ1 junctions and 41 or more independent sequences for Sμ-Sε junctions. At least three mice per genotype in three independent experiments were used for Sμ-Sγ1 junctions and one Ku70−/−HL mouse, two Ku70−/−Lig4−/−HL mice, three Lig4−/−HL, and three WT-HL mice in three independent experiments were used for Sμ-Sε junctions. A detailed summary of all S region junction isolation experiments and mice is presented in Table S4. (C) Percentage of direct Sμ-Sγ1 (left) and Sμ-Sε (right) joins in Ku70−/−HL, Ku70−/−Lig4−/−HL, Lig4−/−HL, XRCC4−/−HL and WT/HL B cells. Ku70 and Ku70Lig4-deficient cells have significantly more direct Sμ-Sγ1 and Sμ-Sε joins than Lig4 germline-deficient cells. *, XRCC4 junctions were acquired and published previously (Yan et al., 2007) and are shown here for comparison. Two-tailed Student’s t test was used for p-value. Data shown are based on 86 or more independent sequences per genotype for Sμ-Sγ1 junctions and 41 or more independent sequences for Sμ-Sε junctions. At least three mice per genotype in three independent experiments were used for Sμ-Sγ1 junctions, and one Ku70−/−HL mouse, two Ku70−/−Lig4−/−HL mice, three Lig4−/−HL, and three WT-HL mice in three independent experiments were used for Sμ-Sε junctions. N, number of independent sequences.
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fig5: The Ku-dependent A-EJ pathway uses a distinct mechanism for S region joining. (a) Classical NHEJ performs both direct (left) and MH-mediated (right) end-joining. Direct joining occurs when DNA ends are joined either immediately, or after processing (removal of overhangs or fill-in by polymerases). MH-mediated joining (right) is facilitated by base-pairing interactions between short stretches of nucleotides at or near the ends of the DSB. (b) MH length in Sμ-Sγ1 (left) and Sμ-Sε (right) junctions from Ku70−/−HL, Ku70−/−Lig4−/−HL, Lig4−/−HL and WT/HL B cells stimulated in culture with αCD40 plus IL-4. Ku70-, Ku70/Lig4-, and Lig4-deficient cells have more Sμ-Sγ1 (left) and Sμ-Sε (right) joins with longer MH than WT. Data shown are based on 86 or more independent sequences per genotype for Sμ-Sγ1 junctions and 41 or more independent sequences for Sμ-Sε junctions. At least three mice per genotype in three independent experiments were used for Sμ-Sγ1 junctions and one Ku70−/−HL mouse, two Ku70−/−Lig4−/−HL mice, three Lig4−/−HL, and three WT-HL mice in three independent experiments were used for Sμ-Sε junctions. A detailed summary of all S region junction isolation experiments and mice is presented in Table S4. (C) Percentage of direct Sμ-Sγ1 (left) and Sμ-Sε (right) joins in Ku70−/−HL, Ku70−/−Lig4−/−HL, Lig4−/−HL, XRCC4−/−HL and WT/HL B cells. Ku70 and Ku70Lig4-deficient cells have significantly more direct Sμ-Sγ1 and Sμ-Sε joins than Lig4 germline-deficient cells. *, XRCC4 junctions were acquired and published previously (Yan et al., 2007) and are shown here for comparison. Two-tailed Student’s t test was used for p-value. Data shown are based on 86 or more independent sequences per genotype for Sμ-Sγ1 junctions and 41 or more independent sequences for Sμ-Sε junctions. At least three mice per genotype in three independent experiments were used for Sμ-Sγ1 junctions, and one Ku70−/−HL mouse, two Ku70−/−Lig4−/−HL mice, three Lig4−/−HL, and three WT-HL mice in three independent experiments were used for Sμ-Sε junctions. N, number of independent sequences.

Mentions: A substantial fraction of CSR junctions from WT B cells are direct, with most of the remainder displaying short MHs; whereas essentially all CSR junctions from XRCC4-deficient B cells display MHs that are on average longer than those from WT B cells (Yan et al., 2007). To further assess the relationship between the A-EJ pathway that generates CSR joins in Ku-deficient cells versus that which occurs in the absence of XRCC4 or Lig4, we sequenced Sμ/Sγ1 and Sμ/Sε junctions from Ku70−/−HL, Ku70−/−Lig4−/−HL, Lig4−/−HL, and WT/HL splenic B cells that were activated with αCD40 plus IL-4. As expected, a substantial percentage of Sμ/Sγ1 and Sμ/Sε junctions from WT/HL B cells were direct (27.1 and 33.3% direct, respectively; Fig. 5, b and c, and Table S4). On the other hand, Sμ/Sγ1 and Sμ/Sε junctions from Lig4−/−HL B cells were almost all MH-mediated (2 and 0% direct, respectively), consistent with the fact that XRCC4 and Lig4 act as a complex in C-NHEJ. Although Sμ/Sγ1 and Sμ/Sε junctions from Ku70−/−HL B cells also displayed increased usage and mean length of MH-mediated joins compared with those from WT/HL B cells (Fig. 5, b and c; Table S4), a substantial fraction were direct (8.9 and 12.2%, respectively; Fig. 5, b and c; Table S4). Similarly, a substantial fraction of Sμ/Sγ1 and Sμ/Sε junctions from Ku70−/−Lig4−/−HL B cells also were direct (18 and 13%, respectively; Fig. 5, b and c, and Table S4). Thus, CSR junctions in Ku-deficient B cells, whether or not Lig4 is present, show a substantial and significant increase in direct joins as compared with junctions generated in the absence of Lig4 (P = 0.04 for Ku70−/−HL; P = 0.003 for Ku70−/−Lig4−/−HL; Fig. 5 C and Table S4). Importantly, the fraction of direct joins is not significantly different between Ku70 versus Ku70 plus Lig4-deficient B cells (P = 0.11 for Sμ/Sγ1 and P = 0.96 for Sμ/Sε junctions; Fig. 5 C), which is consistent with the possibility that another ligase may be predominantly used for CSR end-joining in Ku70−/−HL B cells.


Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4.

Boboila C, Yan C, Wesemann DR, Jankovic M, Wang JH, Manis J, Nussenzweig A, Nussenzweig M, Alt FW - J. Exp. Med. (2010)

The Ku-dependent A-EJ pathway uses a distinct mechanism for S region joining. (a) Classical NHEJ performs both direct (left) and MH-mediated (right) end-joining. Direct joining occurs when DNA ends are joined either immediately, or after processing (removal of overhangs or fill-in by polymerases). MH-mediated joining (right) is facilitated by base-pairing interactions between short stretches of nucleotides at or near the ends of the DSB. (b) MH length in Sμ-Sγ1 (left) and Sμ-Sε (right) junctions from Ku70−/−HL, Ku70−/−Lig4−/−HL, Lig4−/−HL and WT/HL B cells stimulated in culture with αCD40 plus IL-4. Ku70-, Ku70/Lig4-, and Lig4-deficient cells have more Sμ-Sγ1 (left) and Sμ-Sε (right) joins with longer MH than WT. Data shown are based on 86 or more independent sequences per genotype for Sμ-Sγ1 junctions and 41 or more independent sequences for Sμ-Sε junctions. At least three mice per genotype in three independent experiments were used for Sμ-Sγ1 junctions and one Ku70−/−HL mouse, two Ku70−/−Lig4−/−HL mice, three Lig4−/−HL, and three WT-HL mice in three independent experiments were used for Sμ-Sε junctions. A detailed summary of all S region junction isolation experiments and mice is presented in Table S4. (C) Percentage of direct Sμ-Sγ1 (left) and Sμ-Sε (right) joins in Ku70−/−HL, Ku70−/−Lig4−/−HL, Lig4−/−HL, XRCC4−/−HL and WT/HL B cells. Ku70 and Ku70Lig4-deficient cells have significantly more direct Sμ-Sγ1 and Sμ-Sε joins than Lig4 germline-deficient cells. *, XRCC4 junctions were acquired and published previously (Yan et al., 2007) and are shown here for comparison. Two-tailed Student’s t test was used for p-value. Data shown are based on 86 or more independent sequences per genotype for Sμ-Sγ1 junctions and 41 or more independent sequences for Sμ-Sε junctions. At least three mice per genotype in three independent experiments were used for Sμ-Sγ1 junctions, and one Ku70−/−HL mouse, two Ku70−/−Lig4−/−HL mice, three Lig4−/−HL, and three WT-HL mice in three independent experiments were used for Sμ-Sε junctions. N, number of independent sequences.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2822597&req=5

fig5: The Ku-dependent A-EJ pathway uses a distinct mechanism for S region joining. (a) Classical NHEJ performs both direct (left) and MH-mediated (right) end-joining. Direct joining occurs when DNA ends are joined either immediately, or after processing (removal of overhangs or fill-in by polymerases). MH-mediated joining (right) is facilitated by base-pairing interactions between short stretches of nucleotides at or near the ends of the DSB. (b) MH length in Sμ-Sγ1 (left) and Sμ-Sε (right) junctions from Ku70−/−HL, Ku70−/−Lig4−/−HL, Lig4−/−HL and WT/HL B cells stimulated in culture with αCD40 plus IL-4. Ku70-, Ku70/Lig4-, and Lig4-deficient cells have more Sμ-Sγ1 (left) and Sμ-Sε (right) joins with longer MH than WT. Data shown are based on 86 or more independent sequences per genotype for Sμ-Sγ1 junctions and 41 or more independent sequences for Sμ-Sε junctions. At least three mice per genotype in three independent experiments were used for Sμ-Sγ1 junctions and one Ku70−/−HL mouse, two Ku70−/−Lig4−/−HL mice, three Lig4−/−HL, and three WT-HL mice in three independent experiments were used for Sμ-Sε junctions. A detailed summary of all S region junction isolation experiments and mice is presented in Table S4. (C) Percentage of direct Sμ-Sγ1 (left) and Sμ-Sε (right) joins in Ku70−/−HL, Ku70−/−Lig4−/−HL, Lig4−/−HL, XRCC4−/−HL and WT/HL B cells. Ku70 and Ku70Lig4-deficient cells have significantly more direct Sμ-Sγ1 and Sμ-Sε joins than Lig4 germline-deficient cells. *, XRCC4 junctions were acquired and published previously (Yan et al., 2007) and are shown here for comparison. Two-tailed Student’s t test was used for p-value. Data shown are based on 86 or more independent sequences per genotype for Sμ-Sγ1 junctions and 41 or more independent sequences for Sμ-Sε junctions. At least three mice per genotype in three independent experiments were used for Sμ-Sγ1 junctions, and one Ku70−/−HL mouse, two Ku70−/−Lig4−/−HL mice, three Lig4−/−HL, and three WT-HL mice in three independent experiments were used for Sμ-Sε junctions. N, number of independent sequences.
Mentions: A substantial fraction of CSR junctions from WT B cells are direct, with most of the remainder displaying short MHs; whereas essentially all CSR junctions from XRCC4-deficient B cells display MHs that are on average longer than those from WT B cells (Yan et al., 2007). To further assess the relationship between the A-EJ pathway that generates CSR joins in Ku-deficient cells versus that which occurs in the absence of XRCC4 or Lig4, we sequenced Sμ/Sγ1 and Sμ/Sε junctions from Ku70−/−HL, Ku70−/−Lig4−/−HL, Lig4−/−HL, and WT/HL splenic B cells that were activated with αCD40 plus IL-4. As expected, a substantial percentage of Sμ/Sγ1 and Sμ/Sε junctions from WT/HL B cells were direct (27.1 and 33.3% direct, respectively; Fig. 5, b and c, and Table S4). On the other hand, Sμ/Sγ1 and Sμ/Sε junctions from Lig4−/−HL B cells were almost all MH-mediated (2 and 0% direct, respectively), consistent with the fact that XRCC4 and Lig4 act as a complex in C-NHEJ. Although Sμ/Sγ1 and Sμ/Sε junctions from Ku70−/−HL B cells also displayed increased usage and mean length of MH-mediated joins compared with those from WT/HL B cells (Fig. 5, b and c; Table S4), a substantial fraction were direct (8.9 and 12.2%, respectively; Fig. 5, b and c; Table S4). Similarly, a substantial fraction of Sμ/Sγ1 and Sμ/Sε junctions from Ku70−/−Lig4−/−HL B cells also were direct (18 and 13%, respectively; Fig. 5, b and c, and Table S4). Thus, CSR junctions in Ku-deficient B cells, whether or not Lig4 is present, show a substantial and significant increase in direct joins as compared with junctions generated in the absence of Lig4 (P = 0.04 for Ku70−/−HL; P = 0.003 for Ku70−/−Lig4−/−HL; Fig. 5 C and Table S4). Importantly, the fraction of direct joins is not significantly different between Ku70 versus Ku70 plus Lig4-deficient B cells (P = 0.11 for Sμ/Sγ1 and P = 0.96 for Sμ/Sε junctions; Fig. 5 C), which is consistent with the possibility that another ligase may be predominantly used for CSR end-joining in Ku70−/−HL B cells.

Bottom Line: Ku70- or Ku80-deficient B cells have reduced, but still substantial, CSR.Ku-deficient or Ku- plus Lig4-deficient B cells are also biased toward MH-mediated CSR joins; but, in contrast to XRCC4- or Lig4-deficient B cells, generate substantial numbers of direct CSR joins.Our findings suggest that more than one form of A-EJ can function in CSR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The classical nonhomologous end-joining (C-NHEJ) DNA double-strand break (DSB) repair pathway employs the Ku70/80 complex (Ku) for DSB recognition and the XRCC4/DNA ligase 4 (Lig4) complex for ligation. During IgH class switch recombination (CSR) in B lymphocytes, switch (S) region DSBs are joined by C-NHEJ to form junctions either with short microhomologies (MHs; "MH-mediated" joins) or no homologies ("direct" joins). In the absence of XRCC4 or Lig4, substantial CSR occurs via "alternative" end-joining (A-EJ) that generates largely MH-mediated joins. Because upstream C-NHEJ components remain in XRCC4- or Lig4-deficient B cells, residual CSR might be catalyzed by C-NHEJ using a different ligase. To address this, we have assayed for CSR in B cells deficient for Ku70, Ku80, or both Ku70 and Lig4. Ku70- or Ku80-deficient B cells have reduced, but still substantial, CSR. Strikingly, B cells deficient for both Ku plus Lig4 undergo CSR similarly to Ku-deficient B cells, firmly demonstrating that an A-EJ pathway distinct from C-NHEJ can catalyze CSR end-joining. Ku-deficient or Ku- plus Lig4-deficient B cells are also biased toward MH-mediated CSR joins; but, in contrast to XRCC4- or Lig4-deficient B cells, generate substantial numbers of direct CSR joins. Our findings suggest that more than one form of A-EJ can function in CSR.

Show MeSH
Related in: MedlinePlus