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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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Related in: MedlinePlus

The hairpin opening pattern by Artemis:DNA-PKcs for the 17 different coding end hairpins derived from 39 human VH elements. (A) The illustration shows the configuration of coding end hairpin substrates and the cleavage positions by Artemis:DNA-PKcs. The base pairings are indicated as thin lines between the top and bottom strands. The length of the arrows reflects different cleavage efficiency at the four positions (+1 to +4). (B) The hairpin opening products were resolved by denaturing PAGE. The numbers 1–17 correspond to the sequence numbers in Table 1. For each coding end hairpin (HP), there is a set of three lanes. The hairpin opening products (P) were in the middle, with uncut substrate on the left (S), and the hairpin opening markers (M) on the right. For most substrates, the markers correspond to hairpin opening at +1 and +3 positions, except for substrate 11, with only one marker at the +2 position. CE3 substrate appears to show one minor hairpin opening product at greater than the +4 position.
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Figure 4: The hairpin opening pattern by Artemis:DNA-PKcs for the 17 different coding end hairpins derived from 39 human VH elements. (A) The illustration shows the configuration of coding end hairpin substrates and the cleavage positions by Artemis:DNA-PKcs. The base pairings are indicated as thin lines between the top and bottom strands. The length of the arrows reflects different cleavage efficiency at the four positions (+1 to +4). (B) The hairpin opening products were resolved by denaturing PAGE. The numbers 1–17 correspond to the sequence numbers in Table 1. For each coding end hairpin (HP), there is a set of three lanes. The hairpin opening products (P) were in the middle, with uncut substrate on the left (S), and the hairpin opening markers (M) on the right. For most substrates, the markers correspond to hairpin opening at +1 and +3 positions, except for substrate 11, with only one marker at the +2 position. CE3 substrate appears to show one minor hairpin opening product at greater than the +4 position.

Mentions: The terminal 15 bp of the VH elements were used to create the oligont-based cleavage substrates. The name designations for the human VH elements in the second column are those assigned by the Honjo laboratory (19). The first number represents the family and the second number is the individual VH designation. The full sequence for coding end HP 1 (VH 3-13, 3-74 and 6-1) is shown at the top. Each dash for the other sequences indicates the identical nucleotide, while derivations are indicated by the corresponding nucleotides. Hairpin opening positions +1 to +4 indicate the distance 3′ to the hairpin tip, as illustrated in Figure 4A. Hairpin opening efficiency was calculated based on the percentage of total substrate (hairpin opening product/total signal in each lane is shown in Figure 4). The efficiency data shown in the table are averages of two determinations, and the standard deviations are displayed in parentheses below the efficiency. Any product amount less than 0.30% was regarded as no hairpin opening.


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 hairpin opening pattern by Artemis:DNA-PKcs for the 17 different coding end hairpins derived from 39 human VH elements. (A) The illustration shows the configuration of coding end hairpin substrates and the cleavage positions by Artemis:DNA-PKcs. The base pairings are indicated as thin lines between the top and bottom strands. The length of the arrows reflects different cleavage efficiency at the four positions (+1 to +4). (B) The hairpin opening products were resolved by denaturing PAGE. The numbers 1–17 correspond to the sequence numbers in Table 1. For each coding end hairpin (HP), there is a set of three lanes. The hairpin opening products (P) were in the middle, with uncut substrate on the left (S), and the hairpin opening markers (M) on the right. For most substrates, the markers correspond to hairpin opening at +1 and +3 positions, except for substrate 11, with only one marker at the +2 position. CE3 substrate appears to show one minor hairpin opening product at greater than the +4 position.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: The hairpin opening pattern by Artemis:DNA-PKcs for the 17 different coding end hairpins derived from 39 human VH elements. (A) The illustration shows the configuration of coding end hairpin substrates and the cleavage positions by Artemis:DNA-PKcs. The base pairings are indicated as thin lines between the top and bottom strands. The length of the arrows reflects different cleavage efficiency at the four positions (+1 to +4). (B) The hairpin opening products were resolved by denaturing PAGE. The numbers 1–17 correspond to the sequence numbers in Table 1. For each coding end hairpin (HP), there is a set of three lanes. The hairpin opening products (P) were in the middle, with uncut substrate on the left (S), and the hairpin opening markers (M) on the right. For most substrates, the markers correspond to hairpin opening at +1 and +3 positions, except for substrate 11, with only one marker at the +2 position. CE3 substrate appears to show one minor hairpin opening product at greater than the +4 position.
Mentions: The terminal 15 bp of the VH elements were used to create the oligont-based cleavage substrates. The name designations for the human VH elements in the second column are those assigned by the Honjo laboratory (19). The first number represents the family and the second number is the individual VH designation. The full sequence for coding end HP 1 (VH 3-13, 3-74 and 6-1) is shown at the top. Each dash for the other sequences indicates the identical nucleotide, while derivations are indicated by the corresponding nucleotides. Hairpin opening positions +1 to +4 indicate the distance 3′ to the hairpin tip, as illustrated in Figure 4A. Hairpin opening efficiency was calculated based on the percentage of total substrate (hairpin opening product/total signal in each lane is shown in Figure 4). The efficiency data shown in the table are averages of two determinations, and the standard deviations are displayed in parentheses below the efficiency. Any product amount less than 0.30% was regarded as no hairpin opening.

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
Related in: MedlinePlus