A Biochemically Defined System for Coding Joint Formation in V(D)J Recombination

ArticleinMolecular cell 31(4):485-97 · September 2008with18 Reads
DOI: 10.1016/j.molcel.2008.05.029 · Source: PubMed
Abstract
V(D)J recombination is one of the most complex DNA transactions in biology. The RAG complex makes double-stranded breaks adjacent to signal sequences and creates hairpin coding ends. Here, we find that the kinase activity of the Artemis:DNA-PKcs complex can be activated by hairpin DNA ends in cis, thereby allowing the hairpins to be nicked and then to undergo processing and joining by nonhomologous DNA end joining. Based on these insights, we have reconstituted many aspects of the antigen receptor diversification of V(D)J recombination by using 13 highly purified polypeptides, thereby permitting variable domain exon assembly by using this fully defined system in accord with the 12/23 rule for this process. The features of the recombination sites created by this system include all of the features observed in vivo (nucleolytic resection, P nucleotides, and N nucleotide addition), indicating that most, if not all, of the end modification enzymes have been identified.
    • "Oligonucleotides were purified by gel electrophoresis and 5 0 end-labeled where indicated with 32 P by T4 DNA polynucleotide kinase (New England Biolabs). Complementary oligonucleotides were annealed as described previously (Lu et al., 2008). "
    [Show abstract] [Hide abstract] ABSTRACT: During lymphocyte development, the genes encoding antigen receptors are assembled from discrete gene segments by V(D)J recombination. This process is initiated by the proteins RAG-1 and RAG-2, which together cleave DNA at recombination signal sequences (RSSs) that flank the participating gene segments (Gellert, 2002 and Schatz and Swanson, 2011). There are two classes of RSSs, termed 12-RSS and 23-RSS, in which heptamer and nonamer elements are separated by spacers of 12 bp or 23 bp, respectively. DNA cleavage by RAG involves nicking at the junction between the RSS and the coding sequence, followed by transesterification to produce a blunt, 5′-phosphorylated signal end and a coding end that terminates in a hairpin. Under physiologic conditions, cleavage requires the pairing of a 12-RSS with a 23-RSS, so that recombination between like segments is suppressed (Schatz and Swanson, 2011).
    Full-text · Article · Dec 2014
    • "Our results both in cells and with cell extracts show that X4LIG4 regulates DNA-PKcs autophosphorylation. Of note, Lu et al. (2008) have incidentally observed in reconstituted variable (diversity ) joining (V(D)J) recombination reactions that X4LIG4 stimulates DNA-PKcs autophosphorylation mediated by ds DNA oligonucleotides (Lu et al., 2008). In addition, K. Meek and coworkers have also found reduced DNA-PKcs autophosphorylation in X4 defective CHO cells (Meek, K., personal communication). "
    [Show abstract] [Hide abstract] ABSTRACT: Nonhomologous end joining is the primary deoxyribonucleic acid (DNA) double-strand break repair pathway in multicellular eukaryotes. To initiate repair, Ku binds DNA ends and recruits the DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs) forming the holoenzyme. Early end synapsis is associated with kinase autophosphorylation. The XRCC4 (X4)-DNA Ligase IV (LIG4) complex (X4LIG4) executes the final ligation promoted by Cernunnos (Cer)-X4-like factor (XLF). In this paper, using a cell-free system that recapitulates end synapsis and DNA-PKcs autophosphorylation, we found a defect in both activities in human cell extracts lacking LIG4. LIG4 also stimulated the DNA-PKcs autophosphorylation in a reconstitution assay with purified components. We additionally uncovered a kinase autophosphorylation defect in LIG4-defective cells that was corrected by ectopic expression of catalytically dead LIG4. Finally, our data support a contribution of Cer-XLF to this unexpected early role of the ligation complex in end joining. We propose that productive end joining occurs by early formation of a supramolecular entity containing both DNA-PK and X4LIG4-Cer-XLF complexes on DNA ends.
    Full-text · Article · Jan 2013
    • "ATR reinforces this response by the phosphorylation of signaling intermediates including checkpoint kinase 1 (Chk 1) [31]. The most classically defined DNA-PK function is V(D)J recombination that is responsible for antibody diversity and normal immune development (reviewed in [32, 33]). In addition to this well characterized role, however, there is now a significant body of data implicating DNA-PK CS as an upstream element of p53, being involved in the latter's posttranslational modification and apoptotic response to severe DNA damage [20,34353637. "
    [Show abstract] [Hide abstract] ABSTRACT: A key determinant of p53-mediated cell fate following various DNA damage modalities is p21WAF1/CIP1 expression, with elevated p21 expression triggering cell cycle arrest and repressed p21 expression promoting apoptosis. We show that under pro-death DNA damage conditions, the DNA-dependent protein kinase (DNA-PKCS) is recruited to the p21 promoter where it forms a protein complex with p53. The DNA-PKCS-associated p53 displays post-translational modifications that are distinct from those under pro-arrest conditions, ablating p21 transcription and inducing cell death. Inhibition of DNA-PK activity prevents DNA-PKCS binding to p53 on the p21 promoter, restores p21 transcription and significantly reduces cell death. These data demonstrate that DNA-PKCS negatively regulates p21 expression by directly interacting with the p21 transcription machinery via p53, driving the cell towards apoptosis.
    Full-text · Article · Dec 2011
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