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Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways.

Arnal SM, Holub AJ, Salus SS, Roth DB - Nucleic Acids Res. (2010)

Bottom Line: After cleavage, RAG1/2 remain associated with the coding and signal ends (SE) in a post-cleavage complex (PCC), which is critical for their proper joining by classical non-homologous end joining (NHEJ).Since interactions between RAG1/2 and the RSS heptamer element are especially important in forming the RAG-SE complex, we hypothesized that non-consensus heptamer sequences might affect PCC stability.We find that certain non-consensus heptamers, including a cryptic heptamer implicated in oncogenic chromosomal rearrangements, destabilize the PCC, allowing coding and SEs to be repaired by non-standard pathways, including alternative NHEJ.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACT
V(D)J recombination entails double-stranded DNA cleavage at the antigen receptor loci by the RAG1/2 proteins, which recognize conserved recombination signal sequences (RSSs) adjoining variable (V), diversity (D) and joining (J) gene segments. After cleavage, RAG1/2 remain associated with the coding and signal ends (SE) in a post-cleavage complex (PCC), which is critical for their proper joining by classical non-homologous end joining (NHEJ). Certain mutations in RAG1/2 destabilize the PCC, allowing DNA ends to access inappropriate repair pathways such as alternative NHEJ, an error-prone pathway implicated in chromosomal translocations. The PCC is thus thought to discourage aberrant rearrangements by controlling repair pathway choice. Since interactions between RAG1/2 and the RSS heptamer element are especially important in forming the RAG-SE complex, we hypothesized that non-consensus heptamer sequences might affect PCC stability. We find that certain non-consensus heptamers, including a cryptic heptamer implicated in oncogenic chromosomal rearrangements, destabilize the PCC, allowing coding and SEs to be repaired by non-standard pathways, including alternative NHEJ. These data suggest that some non-consensus RSS, frequently present at chromosomal translocations in lymphoid neoplasms, may promote genomic instability by a novel mechanism, disabling the PCC's ability to restrict repair pathway choice.

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A non-consensus heptamer found at the SIL locus allows aberrant repair of CEs. (A) Quantification of FACS data for coding joint formation (n = 5, *P < 0.02). RMP41 cells were transiently transfected with full-length RAG proteins and a fluorescent reporter for coding joint formation, 290-GFP containing either a consensus pair of RSS or substrates with non-consensus heptamers, as indicated. (B) Alternative NHEJ assay (n = 5). RMP41 cells were transiently transfected with full-length RAG proteins using ALT-GFP reporter substrate containing either a consensus pair of RSS or a non-consensus 12RSS, as indicated. Coding joint formation by alternative NHEJ was measured by FACS. (C) Ratio of alternative NHEJ to total NHEJ, normalized to one for a consensus pair of RSS. 12SIL: CACtcTG_ACAAAAACC paired with consensus 23RSS. 12SCL: CACAGcc_ACAAAAACC paired with consensus 23RSS. 12/23SIL: CACtcTG_ACAAAAACC at both RSS. 12/23SCL: CACAGcc_ACAAAAACC at both RSS.
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Figure 5: A non-consensus heptamer found at the SIL locus allows aberrant repair of CEs. (A) Quantification of FACS data for coding joint formation (n = 5, *P < 0.02). RMP41 cells were transiently transfected with full-length RAG proteins and a fluorescent reporter for coding joint formation, 290-GFP containing either a consensus pair of RSS or substrates with non-consensus heptamers, as indicated. (B) Alternative NHEJ assay (n = 5). RMP41 cells were transiently transfected with full-length RAG proteins using ALT-GFP reporter substrate containing either a consensus pair of RSS or a non-consensus 12RSS, as indicated. Coding joint formation by alternative NHEJ was measured by FACS. (C) Ratio of alternative NHEJ to total NHEJ, normalized to one for a consensus pair of RSS. 12SIL: CACtcTG_ACAAAAACC paired with consensus 23RSS. 12SCL: CACAGcc_ACAAAAACC paired with consensus 23RSS. 12/23SIL: CACtcTG_ACAAAAACC at both RSS. 12/23SCL: CACAGcc_ACAAAAACC at both RSS.

Mentions: The cryptic RSS found at the SIL and SCL loci contain multiple changes to the consensus heptamer and nonamer sequences and are thus very poor substrates for V(D)J recombination (35,42). We wondered whether the heptamer variations found at these cryptic RSS, including a fourth and fifth position mutation at the SIL locus (4t5c) and a sixth and seventh position mutation at the SCL locus (6c7c), could destabilize the RAG-SEC and allow repair of breaks at these sites by alternative NHEJ (a consensus nonamer was provided to facilitate cleavage). We found that the 4t5c cryptic heptamer found at the SIL locus almost completely destabilizes the post-cleavage SEC, allowing 70% end release at 37°C (Figure 4A and quantified in Figure 4B, P < 0.02), while modestly decreasing cleavage (to 50% of that observed with a consensus pair of RSS; quantified in Figure 4C) when paired to a consensus 23-RSS. Pairing two 4t5c mutations, one at the 12RSS and one at the 23RSS, resulted in complete end release even at 37°C, allowing cleavage at 40% of wild-type levels (quantified in Figure 4C). A 6c7c heptamer mutation, found at the SCL locus, did not significantly destabilize the SEC (Figure 4A and quantified in Figure 4B) and did not affect cleavage in vitro (Figure 4A and quantified in Figure 4C). As expected, 4t5c and 6c7c both significantly decreased coding joint formation (Figure 5A) (35). Interestingly, the 4t5c, but not the 6c7c cryptic heptamer, increased the level of alternative end joining 4–5-fold (Figure 5B and C). These data suggest that cryptic RSS found the SIL locus may promote joining by error-prone alternative DNA repair pathways.Figure 4.


Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways.

Arnal SM, Holub AJ, Salus SS, Roth DB - Nucleic Acids Res. (2010)

A non-consensus heptamer found at the SIL locus allows aberrant repair of CEs. (A) Quantification of FACS data for coding joint formation (n = 5, *P < 0.02). RMP41 cells were transiently transfected with full-length RAG proteins and a fluorescent reporter for coding joint formation, 290-GFP containing either a consensus pair of RSS or substrates with non-consensus heptamers, as indicated. (B) Alternative NHEJ assay (n = 5). RMP41 cells were transiently transfected with full-length RAG proteins using ALT-GFP reporter substrate containing either a consensus pair of RSS or a non-consensus 12RSS, as indicated. Coding joint formation by alternative NHEJ was measured by FACS. (C) Ratio of alternative NHEJ to total NHEJ, normalized to one for a consensus pair of RSS. 12SIL: CACtcTG_ACAAAAACC paired with consensus 23RSS. 12SCL: CACAGcc_ACAAAAACC paired with consensus 23RSS. 12/23SIL: CACtcTG_ACAAAAACC at both RSS. 12/23SCL: CACAGcc_ACAAAAACC at both RSS.
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Related In: Results  -  Collection

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Figure 5: A non-consensus heptamer found at the SIL locus allows aberrant repair of CEs. (A) Quantification of FACS data for coding joint formation (n = 5, *P < 0.02). RMP41 cells were transiently transfected with full-length RAG proteins and a fluorescent reporter for coding joint formation, 290-GFP containing either a consensus pair of RSS or substrates with non-consensus heptamers, as indicated. (B) Alternative NHEJ assay (n = 5). RMP41 cells were transiently transfected with full-length RAG proteins using ALT-GFP reporter substrate containing either a consensus pair of RSS or a non-consensus 12RSS, as indicated. Coding joint formation by alternative NHEJ was measured by FACS. (C) Ratio of alternative NHEJ to total NHEJ, normalized to one for a consensus pair of RSS. 12SIL: CACtcTG_ACAAAAACC paired with consensus 23RSS. 12SCL: CACAGcc_ACAAAAACC paired with consensus 23RSS. 12/23SIL: CACtcTG_ACAAAAACC at both RSS. 12/23SCL: CACAGcc_ACAAAAACC at both RSS.
Mentions: The cryptic RSS found at the SIL and SCL loci contain multiple changes to the consensus heptamer and nonamer sequences and are thus very poor substrates for V(D)J recombination (35,42). We wondered whether the heptamer variations found at these cryptic RSS, including a fourth and fifth position mutation at the SIL locus (4t5c) and a sixth and seventh position mutation at the SCL locus (6c7c), could destabilize the RAG-SEC and allow repair of breaks at these sites by alternative NHEJ (a consensus nonamer was provided to facilitate cleavage). We found that the 4t5c cryptic heptamer found at the SIL locus almost completely destabilizes the post-cleavage SEC, allowing 70% end release at 37°C (Figure 4A and quantified in Figure 4B, P < 0.02), while modestly decreasing cleavage (to 50% of that observed with a consensus pair of RSS; quantified in Figure 4C) when paired to a consensus 23-RSS. Pairing two 4t5c mutations, one at the 12RSS and one at the 23RSS, resulted in complete end release even at 37°C, allowing cleavage at 40% of wild-type levels (quantified in Figure 4C). A 6c7c heptamer mutation, found at the SCL locus, did not significantly destabilize the SEC (Figure 4A and quantified in Figure 4B) and did not affect cleavage in vitro (Figure 4A and quantified in Figure 4C). As expected, 4t5c and 6c7c both significantly decreased coding joint formation (Figure 5A) (35). Interestingly, the 4t5c, but not the 6c7c cryptic heptamer, increased the level of alternative end joining 4–5-fold (Figure 5B and C). These data suggest that cryptic RSS found the SIL locus may promote joining by error-prone alternative DNA repair pathways.Figure 4.

Bottom Line: After cleavage, RAG1/2 remain associated with the coding and signal ends (SE) in a post-cleavage complex (PCC), which is critical for their proper joining by classical non-homologous end joining (NHEJ).Since interactions between RAG1/2 and the RSS heptamer element are especially important in forming the RAG-SE complex, we hypothesized that non-consensus heptamer sequences might affect PCC stability.We find that certain non-consensus heptamers, including a cryptic heptamer implicated in oncogenic chromosomal rearrangements, destabilize the PCC, allowing coding and SEs to be repaired by non-standard pathways, including alternative NHEJ.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACT
V(D)J recombination entails double-stranded DNA cleavage at the antigen receptor loci by the RAG1/2 proteins, which recognize conserved recombination signal sequences (RSSs) adjoining variable (V), diversity (D) and joining (J) gene segments. After cleavage, RAG1/2 remain associated with the coding and signal ends (SE) in a post-cleavage complex (PCC), which is critical for their proper joining by classical non-homologous end joining (NHEJ). Certain mutations in RAG1/2 destabilize the PCC, allowing DNA ends to access inappropriate repair pathways such as alternative NHEJ, an error-prone pathway implicated in chromosomal translocations. The PCC is thus thought to discourage aberrant rearrangements by controlling repair pathway choice. Since interactions between RAG1/2 and the RSS heptamer element are especially important in forming the RAG-SE complex, we hypothesized that non-consensus heptamer sequences might affect PCC stability. We find that certain non-consensus heptamers, including a cryptic heptamer implicated in oncogenic chromosomal rearrangements, destabilize the PCC, allowing coding and SEs to be repaired by non-standard pathways, including alternative NHEJ. These data suggest that some non-consensus RSS, frequently present at chromosomal translocations in lymphoid neoplasms, may promote genomic instability by a novel mechanism, disabling the PCC's ability to restrict repair pathway choice.

Show MeSH
Related in: MedlinePlus