Ann Orr

DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139

When mammalian cell cultures or mice are exposed to ionizing radiation in survivable or lethal amounts, novel mass components are found in the histone H2A region of two-dimensional gels. Collectively referred to as γ, these components are formed in vivo by several procedures that introduce double-stranded breaks into DNA. γ-Components, which appeared to be the only major novel components detected by mass or 32PO4incorporation on acetic acid-urea-Triton X-100-acetic acid-urea-cetyltrimethylammonium bromide or SDS-acetic acid-urea-cetyltrimethylammonium bromide gels after exposure of cells to ionizing radiation, are shown to be histone H2AX species that have been phosphorylated specifically at serine 139. γ-H2AX appears rapidly after exposure of cell cultures to ionizing radiation; half-maximal amounts are reached by 1 min and maximal amounts by 10 min. At the maximum, approximately 1% of the H2AX becomes γ-phosphorylated per gray of ionizing radiation, a finding that indicates that 35 DNA double-stranded breaks, the number introduced by each gray into the 6 × 109 base pairs of a mammalian G1 genome, leads to the γ-phosphorylation of H2AX distributed over 1% of the chromatin. Thus, about 0.03% of the chromatin appears to be involved per DNA double-stranded break. This value, which corresponds to about 2 × 106 base pairs of DNA per double-stranded break, indicates that large amounts of chromatin are involved with each DNA double-stranded break. Thus, γ-H2AX formation is a rapid and sensitive cellular response to the presence of DNA double-stranded breaks, a response that may provide insight into higher order chromatin structures.

Yeast histone 2A serine 129 is essential for the efficient repair of checkpoint‐blind DNA damage

Cells maintain genomic stability by the coordination of DNA‐damage repair and cell‐cycle checkpoint control. In replicating cells, DNA damage usually activates intra‐S‐phase checkpoint controls, which are characterized by delayed S‐phase progression and increased Rad53 phosphorylation. We show that in budding yeast, the intra‐S‐phase checkpoint controls, although functional, are not activated by the topoisomerase I inhibitor camptothecin (CPT). In a CPT‐hypersensitive mutant strain that lacks the histone 2A (H2A) phosphatidylinositol‐3‐OH kinase (PI(3)K) motif at Ser 129 (h2a‐s129a), the hypersensitivity was found to result from a failure to process full‐length chromosomal DNA molecules during ongoing replication. H2A Ser 129 is not epistatic to the RAD24 and RAD9 checkpoint genes, suggesting a non‐checkpoint role for the H2A PI(3)K site. These results suggest that H2A Ser 129 is an essential component for the efficient repair of DNA double‐stranded breaks (DSBs) during replication in yeast, particularly of those DSBs that do not induce the intra‐S‐phase checkpoint.

Arylisothiocyanate-containing esters of caffeic acid designed as affinity ligands for HIV-1 integrase.

Integrase is an enzyme found in human immunodeficiency virus, which is required for the viral life cycle, yet has no human cellular homologue. For this reason, HIV integrase (IN) has become an important target for the development of new AIDS therapeutics. Irreversible affinity ligands have proven to be valuable tools for studying a number of enzyme and protein systems, yet to date there have been no reports of such affinity ligands for the study of IN. As an initial approach toward irreversible ligand design directed against IN, we appended isothiocyanate functionality onto caffeic acid phenethyl ester (CAPE), a known HIV integrase inhibitor. The choice of isothiocyanate as the reactive functionality, was based on its demonstrated utility in the preparation of affinity ligands directed against a number of other protein targets. Several isomeric CAPE isothiocyanates were prepared to explore the enzyme topography for reactive nitrogen and sulfur nucleophiles vicinal to the enzyme-bound CAPE. The preparation of these CAPE isothiocyanates, required development of new synthetic methodology which employed phenyl thiocarbamates as latent isothiocyanates which could be unmasked near the end of the synthetic sequence. When it was observed that beta-mercaptoethanol (beta-ME), which is required to maintain the catalytic activity of soluble IN (a F185KC280S mutant), reacted with CAPE isothiocyanate functionality to form the corresponding hydroxyethylthiocarbamate, a variety of mutant IN were examined which did not require the presence of beta-ME for catalytic activity. Although in these latter enzymes, CAPE isothiocyanate functionality was presumed to be present and available for acylation by IN nucleophiles, they were equally effective against Cys to Ser mutants. One conclusion of these studies, is that upon binding of CAPE to the integrase, nitrogen or sulfur nucleophiles may not be properly situated in the vicinity of the phenethyl aryl ring to allow reaction with and covalent modification of reactive functionality, such as isothiocyanate groups. The fact that introduction of the isothiocyanate group onto various positions of the phenethyl ring or replacement of the phenyl ring with naphthyl rings, failed to significantly affect inhibitory potency, indicates a degree of insensitivity of this region of the molecule toward structural modification. These findings may be useful in future studies concerned with the development and use of HIV-1 integrase affinity ligands.

Identification of HIV-1 integrase inhibitors based on a four-point pharmacophore.

The rapid emergence of human immunodeficiency virus (HIV) strains resistant to available drugs implies that effective treatment modalities will require the use of a combination of drugs targeting different sites of the HIV life cycle. Because the virus cannot replicate without integration into a host chromosome, HIV-1 integrase (IN) is an attractive therapeutic target. Thus, an effective IN inhibitor should provide additional benefit in combination chemotherapy. A four-point pharmacophore has been identified based on the structures of quinalizarin and purpurin, which were found to be potent IN inhibitors using both a preintegration complex assay and a purified enzyme assay in vitro. Searching with this four-point pharmacophore in the ‘open” part of the National Cancer Institute three-dimensional structure database produced 234 compounds containing the pharmacophore. Sixty of these compounds were tested for their inhibitory activity against IN using the purified enzyme; 19 were found to be active against IN with IC50 values of less than 100 µM, among which 10 had IC50 values of less than 10 µM. These inhibitors can further serve as leads, and studies are in progress to design novel inhibitors based on the results presented in this study.