ATIC, the product of the purH gene, is a 64 kDa bifunctional enzyme that possesses the final two activities in de novo purine biosynthesis, AICAR transformylase and IMP cyclohydrolase. The crystal structure of avian ATIC has been determined to 1.75 A resolution by the MAD method using a Se-methionine modified enzyme. ATIC forms an intertwined dimer with an extensive interface of approximately 5,000 A(2) per monomer. Each monomer is composed of two novel, separate functional domains. The N-terminal domain (up to residue 199) is responsible for the IMPCH activity, whereas the AICAR Tfase activity resides in the C-terminal domain (200-593). The active sites of the IMPCH and AICAR Tfase domains are approximately 50 A apart, with no structural evidence of a tunnel connecting the two active sites. The crystal structure of ATIC provides a framework to probe both catalytic mechanisms and to design specific inhibitors for use in cancer chemotherapy and inflammation.

Protein kinases c-Abl, b-Raf, and p38alpha are recognized as important targets for therapeutic intervention. c-Abl and b-Raf are major targets of marketed oncology drugs Imatinib (Gleevec) and Sorafenib (Nexavar), respectively, and BIRB-796 is a p38alpha inhibitor that reached Phase II clinical trials. A shared feature of these drugs is the fact that they bind to the DFG-out forms of their kinase targets. Although the discovery of this class of kinase inhibitors has increased the level of emphasis on the design of DFG-out inhibitors, the structural determinants for their binding and stabilization of the DFG-out conformation remain unclear. To improve our understanding of these determinants, we determined cocrystal structures of Imatinib and Sorafenib with p38alpha. We also conducted a detailed analysis of Imatinib and Sorafenib binding to p38alpha in comparison with BIRB-796, including binding kinetics, binding interactions, the solvent accessible surface area (SASA) of the ligands, and stabilization of key structural elements of the protein upon ligand binding. Our results yield an improved understanding of the structural requirements for stabilizing the DFG-out form and a rationale for understanding the genesis of ligand selectivity among DFG-out inhibitors of protein kinases.

The histone variant H2AX is rapidly phosphorylated (denoted ␥H2AX) in large chromatin domains (foci) flanking double strand DNA (dsDNA) breaks that are produced by ionizing radiation or genotoxic agents and during V(D)J recombination. H2AX-deficient cells and mice demonstrate increased sensitivity to dsDNA break damage, indicating an active role for ␥H2AX in DNA repair; however, ␥H2AX formation is not required for V(D)J recombination. The latter finding has suggested a greater dependence on ␥H2AX for anchoring free broken ends versus ends that are held together during programmed breakage-joining reactions. Retroviral DNA integration produces a unique intermediate in which a dsDNA break in host DNA is held together by the intervening viral DNA, and such a reaction provides a useful model to distinguish ␥H2AX functions. We found that integration promotes transient formation of ␥H2AX at retroviral integration sites as detected by both immunocytological and chromatin immunoprecipitation methods. These results provide the first direct evidence for the association of newly integrated viral DNA with a protein species that is an established marker for the onset of a DNA damage response. We also show that H2AX is not required for repair of the retroviral integration intermediate as determined by stable transduction. These observations provide independent support for an anchoring model for the function of ␥H2AX in chromatin repair.The evolutionarily conserved histone H2AX comprises approximately 2-25% of the histone H2A pool in mammalian cells and is incorporated randomly into nucleosomes (1). The extended C-terminal tail of H2AX contains a serine (Ser-139) embedded in an invariant SQE motif that is a target for phosphorylation by the phosphatidylinositol 3-kinase-related DNA-PK, ataxia-telangiectasia-mutated (ATM), and ATM and Rad3-related (ATR) protein kinases (2-4). This H2AX serine residue is massively and rapidly phosphorylated at sites of double strand breaks (DSBs) 1 and stalled replication forks (3, 5, 6), forming microscopically visible foci on staining with a specific antibody. This phosphorylation seems to play an important role in processing or repair of DSBs (7,8). H2AX phosphorylation has also been observed at sites of V(D)J recombination (9), meiotic strand breaks, and other physiologically programmed reactions in which DSBs are formed (10 -12).Early events in retroviral replication include entry of the viral capsid with the accompanying enzymes reverse transcriptase and integrase (IN) followed by synthesis of a DNA copy of the viral RNA genome to form a preintegration complex. This complex then enters the nucleus, and integration is first detected at approximately 3-4 h postinfection (13). Retroviral integration is catalyzed by integrase acting on specific sequences at the ends of the viral DNA and via a concerted cleavage-ligation reaction that is mechanistically similar to that catalyzed by RAG proteins during V(D)J recombination (14 -16) (Fig. 1A). As a consequence of integrase-mediated joining, the hos...

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