Mammalian cells repair DNA double-strand breaks (DSBs) through either homologous recombination or non-homologous end joining (NHEJ). V(D)J recombination, a cut-and-paste mechanism for generating diversity in antigen receptors, relies on NHEJ for repairing DSBs introduced by the Rag1-Rag2 protein complex. Animals lacking any of the seven known NHEJ factors are therefore immunodeficient. Nevertheless, DSB repair is not eliminated entirely in these animals: evidence of a third mechanism, 'alternative NHEJ', appears in the form of extremely rare V(D)J junctions and a higher rate of chromosomal translocations. The paucity of these V(D)J events has suggested that alternative NHEJ contributes little to a cell's overall repair capacity, being operative only (and inefficiently) when classical NHEJ fails. Here we find that removing certain portions of murine Rag proteins reveals robust alternative NHEJ activity in NHEJ-deficient cells and some alternative joining activity even in wild-type cells. We propose a two-tier model in which the Rag proteins collaborate with NHEJ factors to preserve genomic integrity during V(D)J recombination.

Monocytic leukemia zinc finger (MOZ)/KAT6A is a MOZ, Ybf2/Sas3, Sas2, Tip60 (MYST)-type histone acetyltransferase that functions as a coactivator for acute myeloid leukemia 1 protein (AML1)-and Ets family transcription factor PU.1-dependent transcription. We previously reported that MOZ directly interacts with p53 and is essential for p53-dependent selective regulation of p21 expression. We show here that MOZ is an acetyltransferase of p53 at K120 and K382 and colocalizes with p53 in promyelocytic leukemia (PML) nuclear bodies following cellular stress. The MOZ-PML-p53 interaction enhances MOZ-mediated acetylation of p53, and this ternary complex enhances p53-dependent p21 expression. Moreover, we identified an Akt/ protein kinase B recognition sequence in the PML-binding domain of MOZ protein. Akt-mediated phosphorylation of MOZ at T369 has a negative effect on complex formation between PML and MOZ. As a result of PML-mediated suppression of Akt, the increased PML-MOZ interaction enhances p21 expression and induces p53-dependent premature senescence upon forced PML expression. Our research demonstrates that MOZ controls p53 acetylation and transcriptional activity via association with PML.protein modification | DNA damage T he p53 protein functions as a key regulator of pathways mediating cellular responses by inducing myriad target genes that regulate diverse cellular processes including cell-cycle arrest, apoptosis, and genomic stability (1-4). Regulation of p53 transcriptional activities is crucial for genotoxic stress because of the varieties of cellular responses that are mediated by p53, which, in some cases, can be mutually exclusive (e.g., arrest and apoptosis) (5). p53 has been detected in discrete nuclear speckles, known as promyelocytic leukemia nuclear bodies (PML-NBs), in which CREB binding protein (CBP)/p300, Tip60, and pRB are also found (6-8).PML was originally identified as a t(15, 17) chromosomal translocation partner with the retinoic acid receptor-α (RARα) in acute promyelocytic leukemia, in which fusion genes encoding the PML-RARα fusion protein are generated (9-11). The ability of PML to interact with activators such as CBP/p300 in the nuclear body suggests that PML could modulate transcription through its ability to stabilize complexes of cofactors (12, 13). Overexpression of PML, γ-irradiation of cells, or oncogenic signals such as Ras overexpression can recruit p53 into PML-NBs (14, 15). The resulting ternary p53-PML-CBP complex then promotes the acetylation of p53 and harmonically coordinates critical tumorsuppressive functions such as apoptosis, senescence, and growth arrest (16,17). Unlike bona fide coactivators such as CBP and p300, PML does not possess intrinsic histone acetylase activity. PML-mediated recruitment of both coactivators promotes p53 modification, such as acetylation and phosphorylation (16,17). Acetylation levels of p53 are known to be significantly enhanced in response to stress and are involved in p53 activation and stabilization (18,19). Given that p300 −/− mouse ...

SUMMARY V(D)J recombination-associated DNA double-strand breaks (DSBs) are normally repaired by the high fidelity, classical non-homologous end joining (cNHEJ) machinery. Previous studies implicated the RAG/DNA post-cleavage complex (PCC) in regulating pathway choice by preventing access to inappropriate repair mechanisms such as homologous recombination (HR) and alternative NHEJ (aNHEJ). Here we report that RAG2’s “acidic hinge”, previously of unknown function, is critical for several key steps. Mutations that reduce the hinge’s negative charge destabilize the PCC, disrupt pathway choice, permit repair of RAG-mediated DSBs by the translocation-prone aNHEJ machinery, and reduce genomic stability in developing lymphocytes. Structural predictions and experimental results support our hypothesis that reduced flexibility of the hinge underlies these outcomes. Furthermore, sequence variants present in the human population reduce the hinge’s negative charge, permit aNHEJ, and diminish genomic integrity.

p23 is an Hsp90-associated protein that regulates signal transduction by the estrogen receptor ␣ (ER); however, the mechanism through which p23 governs ER function remains enigmatic. To obtain a collection of p23 molecules with distinct effects on ER signaling, we screened in yeast a series of random mutations as well as specific sequence alterations based on the p23 crystal structure and further analyzed these mutations for their effect on p23-Hsp90 association in vitro and in vivo. We found that the ability of the p23 mutants to decrease or increase ER signal transduction correlated with their association with Hsp90. We also identified a mutation in the C-terminal tail of p23, which displayed a dominant inhibitory effect on ER transcriptional activation and associates more avidly with Hsp90 relative to the wild type p23. Interestingly, this mutant interacts with Hsp90 in its non-ATP-bound state, whereas the wild type p23 protein interacts exclusively with the ATP-bound form of Hsp90, which may account for its dominant phenotype. In addition, we have uncovered a novel activity of p23 that antagonizes Hsp90 action during times of cell stress. Using molecular modeling and the p23 crystal structure, we found that the p23 mutations affecting ER signaling identified in the screen localized to one face of the molecule, whereas those that had no effect mapped to other parts of the protein. Thus, our structure/function analysis has identified an important regulatory surface on p23 involved in ER signaling and p23 binding to Hsp90.

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.