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

Norris Comprehensive Cancer Center, Room 5428, University of Southern California Keck School of Medicine, 1441 Eastlake Avenue, MC9176, Los Angeles, CA 90089, USA.
Molecular cell (Impact Factor: 14.02). 09/2008; 31(4):485-97. DOI: 10.1016/j.molcel.2008.05.029
Source: PubMed


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.

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    • "Moreover, DNA-PKcs is phosphorylated in living cells at many additional sites [likely more than 40, (18)]; it seems quite likely that autophosphorylation of some sites may occur in cis whereas others occur in trans. In fact, recent studies from Lieber and colleagues (22) as well as Dynan and colleagues (23) using immobilized antibodies or biotin labeled DNA immobilized by streptavidin (respectively) are more consistent with a model whereby DNA-PK is activated (and autophosphorylated) in cis. Although in both studies, care was taken to make sure that DNA-PKcs was not saturating, with studies using antibodies, protein G or streptavidin to immobilize either DNA or DNA-PK, there is the caveat that multiple DNA-PK molecules may be co-localized because of the valence of antibodies, protein G or streptavidin. "
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    ABSTRACT: The DNA-dependent protein kinase (DNA-PK) was identified as an activity and as its three component polypeptides 25 and 15 years ago, respectively. It has been exhaustively characterized as being absolutely dependent on free double stranded DNA ends (to which it is directed by its regulatory subunit, Ku) for its activation as a robust nuclear serine/threonine protein kinase. Here, we report the unexpected finding of robust DNA-PKcs activation by N-terminal constraint, independent of either DNA or its regulatory subunit Ku. These data suggest that an N-terminal conformational change (likely induced by DNA binding) induces enzymatic activation.
    Nucleic Acids Research 12/2011; 40(7):2964-73. DOI:10.1093/nar/gkr1211 · 9.11 Impact Factor
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    • "In vitro experiments using naked DNA indicate that holding of signal ends by RAGs after cleavage is quite tight, whereas holding of coding ends is relatively weak (Agrawal and Schatz, 1997; Jones and Gellert, 2001; Nagawa et al., 2004; Tsai et al., 2002). Consistent with this, in vitro reconstitution of coding joint formation indicates coding end release from the RAG post-cleavage complex, but persistence of RAGs with the signal ends (Lu et al., 2008). These observations raise the possibility that in vivo RAG post-cleavage retention of coding ends occurs largely at the chromatin level. "
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    ABSTRACT: The PHD finger of the RAG2 polypeptide of the RAG1/RAG2 complex binds to the histone H3 modification, trimethylated lysine 4 (H3K4me3), and in some manner increases V(D)J recombination. In the absence of biochemical studies of H3K4me3 on purified RAG enzyme activity, the precise role of H3K4me3 remains unclear. Here, we find that H3K4me3 stimulates purified RAG enzymatic activity at both the nicking (2- to 5-fold) and hairpinning (3- to 11-fold) steps of V(D)J recombination. Remarkably, this stimulation can be achieved with free H3K4me3 peptide (in trans), indicating that H3K4me3 functions via two distinct mechanisms. It not only tethers the RAG enzyme complex to a region of DNA, but it also induces a substantial increase in the catalytic turnover number (k(cat)) of the RAG complex. The H3K4me3 catalytic stimulation applies to suboptimal cryptic RSS sites located at H3K4me3 peaks that are critical in the inception of human T cell acute lymphoblastic lymphomas.
    Molecular cell 07/2009; 34(5):535-44. DOI:10.1016/j.molcel.2009.05.011 · 14.02 Impact Factor
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    ABSTRACT: The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4. We demonstrate here, using in vitro-generated Cernunnos mutants and a series of functional assays in vivo, that the C-terminal region of Cernunnos is dispensable for its activity during DNA repair.
    Molecular and Cellular Biology 03/2009; 29(5):1116-22. DOI:10.1128/MCB.01521-08 · 4.78 Impact Factor
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