The DNA Damage Response: Making It Safe to Play with Knives

The ATM protein kinase, mutations of which are associated with the human disease ataxia-telangiectasia, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer or higher-order multimer, with the kinase domain bound to a region surrounding serine 1981 that is contained within the previously described 'FAT' domain. Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. Most ATM molecules in the cell are rapidly phosphorylated on this site after doses of radiation as low as 0.5 Gy, and binding of a phosphospecific antibody is detectable after the introduction of only a few DNA double-strand breaks in the cell. Activation of the ATM kinase seems to be an initiating event in cellular responses to irradiation, and our data indicate that ATM activation is not dependent on direct binding to DNA strand breaks, but may result from changes in the structure of chromatin.

DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation

During the evolution of cancer, the incipient tumour experiences 'oncogenic stress', which evokes a counter-response to eliminate such hazardous cells. However, the nature of this stress remains elusive, as does the inducible anti-cancer barrier that elicits growth arrest or cell death. Here we show that in clinical specimens from different stages of human tumours of the urinary bladder, breast, lung and colon, the early precursor lesions (but not normal tissues) commonly express markers of an activated DNA damage response. These include phosphorylated kinases ATM and Chk2, and phosphorylated histone H2AX and p53. Similar checkpoint responses were induced in cultured cells upon expression of different oncogenes that deregulate DNA replication. Together with genetic analyses, including a genome-wide assessment of allelic imbalances, our data indicate that early in tumorigenesis (before genomic instability and malignant conversion), human cells activate an ATR/ATM-regulated DNA damage response network that delays or prevents cancer. Mutations compromising this checkpoint, including defects in the ATM-Chk2-p53 pathway, might allow cell proliferation, survival, increased genomic instability and tumour progression.

DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis

The loss of chromosomal integrity from DNA double-strand breaks introduced into mammalian cells by ionizing radiation results in the specific phosphorylation of histone H2AX on serine residue 139, yielding a specific modified form named γ-H2AX. An antibody prepared to the unique region of human γ-H2AX shows that H2AX homologues are phosphorylated not only in irradiated mammalian cells but also in irradiated cells from other species, including Xenopus laevis, Drosophila melanogaster, and Saccharomyces cerevisiae. The antibody reveals that γ-H2AX appears as discrete nuclear foci within 1 min after exposure of cells to ionizing radiation. The numbers of these foci are comparable to the numbers of induced DNA double-strand breaks. When DNA double-strand breaks are introduced into specific partial nuclear volumes of cells by means of a pulsed microbeam laser, γ-H2AX foci form at these sites. In mitotic cells from cultures exposed to nonlethal amounts of ionizing radiation, γ-H2AX foci form band-like structures on chromosome arms and on the end of broken arms. These results offer direct visual confirmation that γ-H2AX forms en masse at chromosomal sites of DNA double-strand breaks. The results further suggest the possible existence of units of higher order chromatin structure involved in monitoring DNA integrity.

/pdf/megabase-chromatin-domains-involved-in-dna-double-strand-1pw1vrkkae.pdf

Megabase chromatin domains involved in DNA double-strand breaks in vivo.

A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage.

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

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.

/pdf/yeast-histone-2a-serine-129-is-essential-for-the-efficient-5fdciipmtt.pdf

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

Several classes of antitumor drugs are known to stabilize topoisomerase complexes in which the enzyme is covalently bound to a terminus of a DNA strand break. The DNA cleavage sites generally are different for each class of drugs. We have determined the DNA sequence locations of a large number of drug-stimulated cleavage sites of topoisomerase II, and find that the results provide a clue to the possible structure of the complexes and the origin of the drug-specific differences. Cleavage enhancements by VM-26 and amsacrine (m-AMSA), which are representative of different classes of topoisomerase II inhibitors, have strong dependence on bases directly at the sites of cleavage. The preferred bases were C at the 3' terminus for VM-26 and A at the 5' terminus for m-AMSA. Also, a region of dyad symmetry of 12 to 16 base pairs was detected about the enzyme cleavage positions. These results are consistent with those obtained with doxorubicin, although in the case of doxorubicin, cleavage requires the presence of an A at the 3' terminus of at least one the pair of breaks that constitute a double-strand cleavage (Capranico et al., Nucleic Acids Res., 1990, 18: 6611). These findings suggest that topoisomerase II inhibitors may stack with one or the other base pair flanking the enzyme cleavage sites.

Local base sequence preferences for DNA cleavage by mammalian topoisomerase II in the presence of amsacrine or teniposide

Human immunodeficiency virus type 1 integrase (HIV-1 IN) is an essential enzyme for effective viral replication. Therefore, IN inhibitors are being sought for chemotherapy against AIDS. We had previously identified a series of salicylhydrazides as potent inhibitors of IN in vitro (Neamati, N.; et al. J. Med. Chem. 1998, 41, 3202−3209.). Herein, we report the design, synthesis, and antiviral activity of three novel mercaptosalicylhydrazide (MSH) derivatives. MSHs were effective against the IN catalytic core domain and inhibited IN binding to HIV LTR DNA. They also inhibited catalytic activities of IN in IN−DNA preassembled complexes. Site-directed mutagenesis and molecular modeling studies suggest that MSHs bind to cysteine 65 and chelate Mg2+ at the active site of HIV-1 IN. Contrary to salicylhydrazides, the MSHs are 300-fold less cytotoxic and exhibit antiviral activity. They are also active in Mg2+-based assays, while IN inhibition by salicylhydrazides is strictly Mn2+-dependent. Additionally, in target...

Metal-dependent inhibition of HIV-1 integrase.

Azatoxin [NSC 640737-M; 5.R,11aS-1H,6H,3-one-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-hydroxyphenyl) oxazolo (3′,4′:1,6)pyrido-(3,4-b)indole] was rationally designed from a model for the pharmacophore of drugs with topoisomerase II inhibition activity. This pharmacophore has at least 2 domains: a quasiplanar polycyclic ring system proposed to bind between the DNA base pairs and a pendant substituent proposed to interact with the enzyme and/or to the DNA grooves. The present study shows that, in cell free systems, azatoxin induces a large number of double strand-breaks in linear Simian virus 40 and human c-myc DNA. These breaks yield cleavage patterns that are different from those of well established topoisomerase II inhibitors (epipodophyllotoxins, amsacrine, mitoxantrone). Azatoxin also inhibits the catalytic activity of purified topoisomerase II, and is a nonintercalator. The structure-activity relationship of 3 isomers and 6 derivatives of azatoxin shows a stringent stereochemical requirement for activity. The effects of azatoxin pendant ring substitution on topoisomerase II mediated DNA cleavage activity were similar to the relationship observed for etoposide.

https://cancerres.aacrjournals.org/content/canres/52/16/4478.full.pdf

Ann Orr

Papers

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

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

Local base sequence preferences for DNA cleavage by mammalian topoisomerase II in the presence of amsacrine or teniposide

Metal-dependent inhibition of HIV-1 integrase.

Rational design and molecular effects of a new topoisomerase II inhibitor, azatoxin.