DNA‐dependent protein kinase (DNA‐PK or the scid factor) and Ku are critical for DNA end‐joining in V(D)J recombination and in general non‐homologous double‐strand break repair. One model for the function of DNA‐PK is that it forms a complex with Ku70/86, and this complex then binds to DNA ends, with Ku serving as the DNA‐binding subunit. We find that DNA‐PK can itself bind to linear DNA fragments ranging in size from 18 to 841 bp double‐stranded (ds) DNA, as indicated by: (i) mobility shifts; (ii) crosslinking between the DNA and DNA‐PK; and (iii) atomic‐force microscopy. Binding of the 18 bp ds DNA to DNA‐PK activates it for phosphorylation of protein targets, and this level of activation is not increased by addition of purified Ku70/86. Ku can stimulate DNA‐PK activity beyond this level only when the DNA fragments are long enough for the independent binding to the DNA of both DNA‐PK and Ku. Atomic‐force microscopy indicates that under such conditions, the DNA‐PK binds at the DNA termini, and Ku70/86 assumes a position along the ds DNA that is adjacent to the DNA‐PK.

Eighteen histone deacetylases (HDACs) are present in humans, categorized into two groups: zinc-dependent enzymes (HDAC1-11) and NAD ؉ -dependent enzymes (sirtuins 1-7). Among zinc-dependent HDACs, HDAC6 is unique. It has a cytoplasmic localization, two catalytic sites, a ubiquitin-binding site, and it selectively deacetylases ␣-tubulin and Hsp90. Here, we report the discovery that the redox regulatory proteins, peroxiredoxin (Prx) I and Prx II are specific targets of HDAC6. Prx are antioxidants enzymes whose main function is H2O2 reduction. Prx are elevated in many cancers and neurodegenerative diseases. The acetylated form of Prx accumulates in the absence of an active HDAC6. Acetylation of Prx increases its reducing activity, its resistance to superoxidation, and its resistance to transition to highmolecular-mass complexes. Thus, HDAC6 and Prx are targets for modulating intracellular redox status in therapeutic strategies for disorders as disparate as cancers and neurodegenerative diseases.acetylation ͉ hydrogen peroxide ͉ histone deacetylase inhibitors

DNA-dependent protein kinase (DNA-PK) is the only eukaryotic protein kinase known to be specifically activated by double-stranded DNA (dsDNA) termini, accounting for its importance in repair of dsDNA breaks and its role in physiologic processes involving dsDNA breaks, such as V(D)J recombination. In this study we conducted kinase and binding analyses using DNA-PK on DNA termini of various lengths in the presence and absence of Ku. We confirmed our previous observations that DNA-PK can bind DNA termini in the absence of Ku, and we determined rate constants for binding. However, in the presence of Ku, DNA-PK can assume either a productive or a nonproductive configuration, depending on the length of the DNA terminus. For dsDNA greater than 26 bp, the productive mode is achieved and Ku increases the affinity of the DNA-PK for the Ku:DNA complex. The change in affinity is achieved by increases in both the kinetic association rate and reduction in the kinetic dissociation rate. For dsDNA smaller than 26 bp, the nonproductive mode, in which DNA-PK is bound to Ku:DNA but is inactive as a kinase, is assumed. Both the productive and nonproductive configurations are likely to be of physiologic importance, depending on the distance of the dsDNA break site to other protein complexes, such as nucleosomes.One can broadly classify eukaryotic DNA repair into excision repair, mismatch repair, and double-strand break repair. Two types of double-strand break repair are homologous recombination and nonhomologous DNA end joining (NHEJ). NHEJ is the major mechanism of repairing double-stranded DNA (dsDNA) breaks during most of the cell cycle yet is the least understood type of DNA repair. Unlike single-strand breaks, which have the other strand to maintain both the physical integrity and the information content of the DNA, doublestrand breaks do not. Hence, double-strand breaks have the highest potential to result in either loss of genetic information or loss of chromosomal integrity, each of which can further contribute to subsequent genomic destabilization events.The nucleic acid and protein biochemical properties of NHEJ are largely undefined (28). Recently, we and others determined that DNA ligase IV complex is responsible for NHEJ in Saccharomyces cerevisiae (34,39,40). In mammalian cells, we and others also recently identified a complex of XRCC4 (X-ray cross-complementation group 4) and DNA ligase IV (9, 17). The XRCC4 stimulates the DNA ligase IV by physical association (17). This is interesting because null mutations in XRCC4 in mammalian cells result in sensitivity to ionizing radiation, especially in G 1 and early S phases of the cell cycle (15). XRCC4 mutant cells that are made active for V(D)J recombination (by transfection with RAG expression vectors) are defective for both signal and coding joint formation (32,37,38). All of the XRCC mutants that are defective for double-strand break repair are also defective for V(D)J recombination (28). This is not surprising given that V(D

Reversible protein phosphorylation is a key regulatory process in all living cells. Deregulation of modification control mechanisms, especially in the case of tyrosine, may lead to malignant transformation and disease. Phosphotyrosine (p-Tyr) accounts for only 0.05% of the total cellular phospho-amino acid content, yet plays an unusually prominent role in eukaryotic signaling, development, and growth. Tracking temporal and positional p-Tyr changes across the cellular proteome, i.e. tyrosine phosphoproteomics, is therefore tremendously valuable. Here, we describe and evaluate a prototype antibody (Ab) microarray platform to monitor changes in protein Tyr phosphorylation. Availability permitting, a virtually unlimited number of Abs, each recognizing a specific cellular protein, may be arrayed on a chip, incubated with total cell or tissue extracts or with biological fluids, and then probed with a fluorescently labeled p-Tyr-specific monoclonal Ab, PY-KD1, specifically generated for this assay as part of the current study. The optimized protocol allowed detection of changes in the Tyr phosphorylation state of selected proteins using submicrogram to low nanogram of total protein extract, amounts that may conceivably be obtained from a thousand to a hundred thousand cells, or less, depending on the cell or tissue type. The assay platform was evaluated by assessing changes in a rationally selected subset of the Tyr phosphoproteome of BcrAbl-expressing cells treated with a specific inhibitor, Gleevec, and of epidermal growth factor (EGF)-treated HeLa cells. The results, ratiometric rather than strictly quantitative in nature, conformed with previous identifications of several Bcr-Abl and EGF receptor targets, and associated proteins, as detected by exhaustive mass spectrometric analyses. The Ab microarray method described here offers advantages of low sample and reagent consumption, scalability, detection multiplexing, and potential compatibility with microfluidic devices and automation. The system may hold particular promise for dissecting signaling pathways, molecular classification of tumors, and profiling of novel target-cancer drugs.

Histone modification regulates gene expression, and one major regulatory step in this process is the ability of proteins that recognize epigenetic marks to recruit enzymes required to specify transcriptional outcome. Here we show that BRD7 is a component of hSWI–SNF complexes that interacts with PRMT5 and PRC2. Recruitment studies revealed that BRD7 co-localizes with PRMT5 and PRC2 on ‘suppressor of tumorigenecity 7’ (ST7) and retinoblastoma-like protein 2 (RBL2) promoters in patient-derived B cell lines, and that its association with these target genes correlates with hypermethylation of H3R8, H4R3 and H3K27. Furthermore, inhibition of BRD7 expression reduces PRMT5 and PRC2 recruitment to ST7 and RBL2 promoters; however, only ST7 becomes transcriptionally derepressed. Evaluation of the PRMT5- and PRC2-induced epigenetic marks revealed that while H3(Me2)R8, H4(Me2)R3 and H3(Me3)K27 marks are erased from the ST7 promoter, demethylation of RBL2 promoter histones is incomplete. We also show that the arginine demethylase (RDM) JMJD6, which can erase PRMT5-induced H4R3 methylation, and the H3K27-lysine-specific demethylases, KDM6A/UTX and KDM6B/JMJD3, are differentially recruited to ST7 and RBL2. These findings highlight the role played by BRD7 in PRMT5- and PRC2-induced transcriptional silencing, and indicate that recruitment of specific RDMs and KDMs is required for efficient transcriptional derepression.