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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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Hairpin opening by Artemis:DNA-PKcs as a function of KCl or Ku. (A) The substrate used here is coding end hairpin 1 (CE1). Proteins in the reactions are indicated above the corresponding lanes. Reactions 1–4 are done in 25 mM KCl, while reactions 5–8 are done in 75 mM KCl. To activate DNA-PKcs, reactions 1–7 included a 60 bp double-strand DNA with short overhangs, whereas reaction 8 instead contains a pseudo-Y structured DNA with a 20 bp double-strand region as depicted. The numbers along the bottom are the quantitation of hairpin opening (product/total). (B) Radioactive kinase assays with DNA-PKcs and Artemis were carried out at the indicated KCl concentrations, and Ku protein was added to reaction 5.
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Figure 3: Hairpin opening by Artemis:DNA-PKcs as a function of KCl or Ku. (A) The substrate used here is coding end hairpin 1 (CE1). Proteins in the reactions are indicated above the corresponding lanes. Reactions 1–4 are done in 25 mM KCl, while reactions 5–8 are done in 75 mM KCl. To activate DNA-PKcs, reactions 1–7 included a 60 bp double-strand DNA with short overhangs, whereas reaction 8 instead contains a pseudo-Y structured DNA with a 20 bp double-strand region as depicted. The numbers along the bottom are the quantitation of hairpin opening (product/total). (B) Radioactive kinase assays with DNA-PKcs and Artemis were carried out at the indicated KCl concentrations, and Ku protein was added to reaction 5.

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)

Hairpin opening by Artemis:DNA-PKcs as a function of KCl or Ku. (A) The substrate used here is coding end hairpin 1 (CE1). Proteins in the reactions are indicated above the corresponding lanes. Reactions 1–4 are done in 25 mM KCl, while reactions 5–8 are done in 75 mM KCl. To activate DNA-PKcs, reactions 1–7 included a 60 bp double-strand DNA with short overhangs, whereas reaction 8 instead contains a pseudo-Y structured DNA with a 20 bp double-strand region as depicted. The numbers along the bottom are the quantitation of hairpin opening (product/total). (B) Radioactive kinase assays with DNA-PKcs and Artemis were carried out at the indicated KCl concentrations, and Ku protein was added to reaction 5.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175297&req=5

Figure 3: Hairpin opening by Artemis:DNA-PKcs as a function of KCl or Ku. (A) The substrate used here is coding end hairpin 1 (CE1). Proteins in the reactions are indicated above the corresponding lanes. Reactions 1–4 are done in 25 mM KCl, while reactions 5–8 are done in 75 mM KCl. To activate DNA-PKcs, reactions 1–7 included a 60 bp double-strand DNA with short overhangs, whereas reaction 8 instead contains a pseudo-Y structured DNA with a 20 bp double-strand region as depicted. The numbers along the bottom are the quantitation of hairpin opening (product/total). (B) Radioactive kinase assays with DNA-PKcs and Artemis were carried out at the indicated KCl concentrations, and Ku protein was added to reaction 5.
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