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Extent to which hairpin opening by the Artemis:DNA-PKcs complex can contribute to junctional diversity in V(D)J recombination.

Lu H, Schwarz K, Lieber MR - Nucleic Acids Res. (2007)

Bottom Line: V(D)J recombination events are initiated by cleavage at gene segments by the RAG1:RAG2 complex, which results in hairpin formation at the coding ends.The hairpin opening position varies over the region from 1 to 4 nt 3' of the hairpin tip, leading to a 2-8 nt single-stranded 3' overhang at each coding end.This information provides greater clarity on the extent to which the hairpin opening position contributes to junctional diversification in V(D)J recombination.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Norris Comprehensive Cancer Center, Los Angeles, CA, USA.

ABSTRACT
V(D)J recombination events are initiated by cleavage at gene segments by the RAG1:RAG2 complex, which results in hairpin formation at the coding ends. The hairpins are opened by the Artemis:DNA-PKcs complex, and then joined via the nonhomologous DNA end joining (NHEJ) process. Here we examine the opening of the hairpinned coding ends from all of the 39 functional human V(H) elements. We find that there is some sequence-dependent variation in the efficiency and even the position of hairpin opening by Artemis:DNA-PKcs. The hairpin opening efficiency varies over a 7-fold range. The hairpin opening position varies over the region from 1 to 4 nt 3' of the hairpin tip, leading to a 2-8 nt single-stranded 3' overhang at each coding end. This information provides greater clarity on the extent to which the hairpin opening position contributes to junctional diversification in V(D)J recombination.

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The endonuclease activity of Artemis:DNA-PKcs is linear over a 90-min time course. (A) Two different coding end hairpins 1 and 9 (CE1 and CE9) were assayed for time courses of hairpin opening by Artemis:DNA-PKcs. The arrowheads point to the primary hairpin opening products at the +2 position. (B) The major products at each time point were quantified and normalized to the product at the last time point (90 min). A linear trend line was added and the R-squared value was displayed on the chart. (C) The time course of 3′ DNA overhang cleavage by Artemis:DNA-PKcs. The 3′ DNA overhang substrate was illustrated on the top. The major products are identified using arrows with the nt sizes and corresponding cleavage positions (arrowheads) depicted on the right. (D) The primary cleavage products of 26 nt at each time point were plotted in the same manner as in (B).
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Figure 1: The endonuclease activity of Artemis:DNA-PKcs is linear over a 90-min time course. (A) Two different coding end hairpins 1 and 9 (CE1 and CE9) were assayed for time courses of hairpin opening by Artemis:DNA-PKcs. The arrowheads point to the primary hairpin opening products at the +2 position. (B) The major products at each time point were quantified and normalized to the product at the last time point (90 min). A linear trend line was added and the R-squared value was displayed on the chart. (C) The time course of 3′ DNA overhang cleavage by Artemis:DNA-PKcs. The 3′ DNA overhang substrate was illustrated on the top. The major products are identified using arrows with the nt sizes and corresponding cleavage positions (arrowheads) depicted on the right. (D) The primary cleavage products of 26 nt at each time point were plotted in the same manner as in (B).

Mentions: The oligonucleotides (oligonts) used for the hairpin opening in Figure 1A are shown in Table 1. The oligonts used to generate the 3′ overhang substrate in Figure 1C are YM-149 and YM-68 [sequences were described in (18)]. The sequence for each coding end hairpin substrate is depicted in Figure 3A: 5′-GGGCCC-CE-CE anti-parallel-CCCGGG-3′, and the sequences for CE (coding end) are shown in Table 1. The human VH names in Table 1 are according to the Honjo laboratory (19). For activation of DNA-PKcs, the 60 bp double-strand DNA was annealed form oligonts YM-74 and YM-75 [sequences in (7)]. The pseudo-Y DNA was annealed from oligonts YM-220 and YM-221 [sequences in (20)].Figure 1.


Extent to which hairpin opening by the Artemis:DNA-PKcs complex can contribute to junctional diversity in V(D)J recombination.

Lu H, Schwarz K, Lieber MR - Nucleic Acids Res. (2007)

The endonuclease activity of Artemis:DNA-PKcs is linear over a 90-min time course. (A) Two different coding end hairpins 1 and 9 (CE1 and CE9) were assayed for time courses of hairpin opening by Artemis:DNA-PKcs. The arrowheads point to the primary hairpin opening products at the +2 position. (B) The major products at each time point were quantified and normalized to the product at the last time point (90 min). A linear trend line was added and the R-squared value was displayed on the chart. (C) The time course of 3′ DNA overhang cleavage by Artemis:DNA-PKcs. The 3′ DNA overhang substrate was illustrated on the top. The major products are identified using arrows with the nt sizes and corresponding cleavage positions (arrowheads) depicted on the right. (D) The primary cleavage products of 26 nt at each time point were plotted in the same manner as in (B).
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Related In: Results  -  Collection

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Figure 1: The endonuclease activity of Artemis:DNA-PKcs is linear over a 90-min time course. (A) Two different coding end hairpins 1 and 9 (CE1 and CE9) were assayed for time courses of hairpin opening by Artemis:DNA-PKcs. The arrowheads point to the primary hairpin opening products at the +2 position. (B) The major products at each time point were quantified and normalized to the product at the last time point (90 min). A linear trend line was added and the R-squared value was displayed on the chart. (C) The time course of 3′ DNA overhang cleavage by Artemis:DNA-PKcs. The 3′ DNA overhang substrate was illustrated on the top. The major products are identified using arrows with the nt sizes and corresponding cleavage positions (arrowheads) depicted on the right. (D) The primary cleavage products of 26 nt at each time point were plotted in the same manner as in (B).
Mentions: The oligonucleotides (oligonts) used for the hairpin opening in Figure 1A are shown in Table 1. The oligonts used to generate the 3′ overhang substrate in Figure 1C are YM-149 and YM-68 [sequences were described in (18)]. The sequence for each coding end hairpin substrate is depicted in Figure 3A: 5′-GGGCCC-CE-CE anti-parallel-CCCGGG-3′, and the sequences for CE (coding end) are shown in Table 1. The human VH names in Table 1 are according to the Honjo laboratory (19). For activation of DNA-PKcs, the 60 bp double-strand DNA was annealed form oligonts YM-74 and YM-75 [sequences in (7)]. The pseudo-Y DNA was annealed from oligonts YM-220 and YM-221 [sequences in (20)].Figure 1.

Bottom Line: V(D)J recombination events are initiated by cleavage at gene segments by the RAG1:RAG2 complex, which results in hairpin formation at the coding ends.The hairpin opening position varies over the region from 1 to 4 nt 3' of the hairpin tip, leading to a 2-8 nt single-stranded 3' overhang at each coding end.This information provides greater clarity on the extent to which the hairpin opening position contributes to junctional diversification in V(D)J recombination.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Norris Comprehensive Cancer Center, Los Angeles, CA, USA.

ABSTRACT
V(D)J recombination events are initiated by cleavage at gene segments by the RAG1:RAG2 complex, which results in hairpin formation at the coding ends. The hairpins are opened by the Artemis:DNA-PKcs complex, and then joined via the nonhomologous DNA end joining (NHEJ) process. Here we examine the opening of the hairpinned coding ends from all of the 39 functional human V(H) elements. We find that there is some sequence-dependent variation in the efficiency and even the position of hairpin opening by Artemis:DNA-PKcs. The hairpin opening efficiency varies over a 7-fold range. The hairpin opening position varies over the region from 1 to 4 nt 3' of the hairpin tip, leading to a 2-8 nt single-stranded 3' overhang at each coding end. This information provides greater clarity on the extent to which the hairpin opening position contributes to junctional diversification in V(D)J recombination.

Show MeSH