Volume 383, Issue 6

The induction of doublestrand breaks (DSBs) in DNA by exposure to DNA damaging agents, or as intermediates in normal cellular processes, constitutes a severe threat for the integrity of the genome. If not properly repaired, DSBs may result in chromosomal aberrations, which, in turn, can lead to cell death or to uncontrolled cell growth. To maintain the integrity of the genome, multiple pathways for the repair of DSBs have evolved during evolution: homologous recombination (HR), nonhomologous end joining (NHEJ) and singlestrand annealing (SSA). HR has the potential to lead to accurate repair of DSBs, whereas NHEJ and SSA are essentially mutagenic. In yeast, DSBs are primarily repaired via highfidelity repair of DSBs mediated by HR, whereas in higher eukaryotes, both HR and NHEJ are important. In this review, we focus on the functional conservation of HR from fungi to mammals and on the role of the individual proteins in this process.

DNA methylation at position C5 of the pyrimidine ring of cytosine in mammalian genomes has received a great deal of research interest due to its importance in many biological phenomena. It is associated with events such as epigenetic gene silencing and the maintenance of genome integrity. Aberrant DNA methylation, particularly that of chromosomal regions called CpG islands, is an important step in carcinogenesis. In order to elucidate methylation profiling of complex genomes, various methods have been developed. Many of these methods are based on the differential reactivity of cytosine and 5-methylcytosine to various chemicals. The combined use of these chemical reactions and other preexisting methods has enabled the discrimination of cytosine and 5- methylcytosine in complex genomes. The use of proteins that preferentially bind to methylated DNA has also successfully been used to discriminate between methylated and unmethylated sites. The chemical and structural dissection of the in vivo processes of enzymatic methylation and the binding of methyl CpG binding proteins provides evidence for the complex mechanisms that nature has acquired. In this review we summarize the methods available for the discrimination between cytosine and 5-methylcytosine in complex genomes.

Loss of genetic material from chromosome 3p21.3 is one of the most common and earliest events in the pathogenesis of lung cancer and many other solid tumors. The chromosomal area 3p21.3 is thought to harbor at least one important tumor suppressor gene, which, despite many years of investigation, has remained elusive. In our previous studies, we have identified and cloned a gene from the common homozygous deletion area at 3p21.3. The gene, named RASSF1A (Ras ASSociation domain Family 1A), has homology to a mammalian Ras effector. The RASSF1A gene is epigenetically inactivated in a large percentage of human lung cancers, in particular small cell carcinomas. A high frequency of methylation of RASSF1A is found also in breast cancers, renal cell carcinomas, ovarian, gastric and bladder cancers, and in neuroblastomas. The RASSF1A gene is a candidate for a tumor suppressor gene in 3p21.3.

Molecular pathways to colorectal cancer involve multiple genetic changes, whereby extensive oxyradical damage causes mutations in cancerrelated genes and leads to a cycle of cell death and regeneration. Besides direct oxidative DNAdamage, reactive oxygen and nitrogen species can induce etheno ()DNA adducts mainly via trans-4-hydroxy-2-nonenal, generated as the major aldehyde by lipid peroxidation (LPO) of ω-6 PUFAs. Patients with familial adenomatous polyposis (FAP) develop multiple colorectal adenomas. In affected tissues increased LPO could be triggered due to increased arachidonic acid metabolism as a result of elevated cyclooxygenases. Our studies demonstrated an increased DNA adduct level in affected colon epithelia of FAP patients. DNA adducts are promutagenic and can cause genomic instability that drives colorectal adenoma to malignancy. We have further investigated the potential chemopreventive properties of olive oil and its polyphenolic components. Mediterranean diet, of which olive oil is a major fatty acid source, has protective effects against human breast and colorectal cancers. Olive oil extracts and the newly identified lignan fractions showed high antioxidant capacity in vitro. As DNA adducts are biomarkers for oxidative stress and LPO induced DNA damage, they can verify the efficacy of newly identified antioxidants, e.g. from olive oil, as chemopreventive agents against colon carcinogenesis.

Cancer arises as a result of several factors, including multiple genes and environmental exposures. It is generally accepted that genetic polymorphisms are associated with most common disorders like cancer. The majority of polymorphisms are single nucleotide polymorphisms (SNPs) which occur with a frequency of 10 exp.(-6). Susceptibilityconferring alleles are not sufficient to cause disease, but modulate the risk in combination with other alleles and environmental exposures, except in the extreme case of Mendelian cancer syndromes (e.g. FAP, HNPCC, Rb). The Environmental Genome Project identifies, among others, two lines of research along which we have been working and are the topic of the present paper, namely (i) alleledisease associations and (ii) functional studies of allelic variants. Casecontrol association studies conducted by us and others showed that polymorphism at a single site could increase riskpredictability by a factor < 2. It is known, however, that the individual risk predictability increases by associating multiple genetic polymorphisms as was demonstrated for breast, renal and thyroid cancer. Functional genomics of the putative susceptibilityalleles involved in cancers can improve substantially the strength of association studies. This calls for cellsystems capable of tracking different gene activities, which may clarify the possible role of allelic variants in certain cancers. This endeavour is likely to be met by the bacterial tester strain, MTC, described here.

This review discusses recent aspects of oxidation reactions of DNA and model compounds involving mostly .OH radicals, oneelectron transfer process and singlet oxygen ([1]O[2]). Emphasis is placed on the formation of double DNA lesions involving a purine base on one hand and either a pyrimidine base or a 2-deoxyribose moiety on the other hand. Structural and mechanistic information is also provided on secondary oxidation reactions of 8-oxo-7,8-dihydro-2deoxyguanosine (8- oxodGuo), a major DNA marker of oxidative stress. Another major topic which is addressed here deals with recent developments in the measurement of oxidative base damage to cellular DNA. This has been mostly achieved using the accurate and highly specific HPLC method coupled with the tandem mass spectrometry detection technique. Interestingly, optimized conditions of DNA extraction and subsequent workup allow the accurate measurement of 11 modified nucleosides and bases within cellular DNA upon exposure to oxidizing agents, including UVA and ionizing radiations. In addition, the modified comet assay, which involves the use of bacterial DNA Nglycosylases to reveal two main classes of oxidative base damage, is applicable to isolated cells and is particularly suitable when only small amounts of biological material are available. Finally, recently available data on the substrate specificity of DNA repair enzymes belonging to the base excision pathways are briefly reviewed.

The small DNA binding protein HNS is attracting broad interest for its profound involvement in the regulation of bacterial physiology. It is involved in the regulation of many genes in response to a changing environment and functions in the adaptation to many different kinds of stress. Many HNScontrolled genes, including the hns gene itself, are further linked to global regulatory networks. HNS thus plays a key role in maintaining bacterial homeostasis under conditions of a rapidly changing environment. In this review we summarize recent results from combined biochemical and biophysical efforts which have yielded new insights into the threedimensional structure and function of HNS. The protein consists of two distinct domains separated by an unstructured linker region, and the structural details available today have helped to understand how these domains may interact with each other or with ligand molecules. Functional studies have, in addition, revealed mechanistic clues for the various HNS activities, like temperature or growth phasedependent regulation. Important elements for the specific regulatory activities of HNS comprise different modes of DNA binding, protein oligomerization, the competition with other regulators and the fact that the topology of the target DNA is modulated during complex formation. The distinctive ability to recognize nucleic acid structures in combination with other proteins also explains HNSdependent posttranscriptional activities where the interaction with defined RNA structures and the interference with RNA/protein complexes during mRNA translation are crucial for regulation. Thus, protein/protein interactions, in combination with the recognition and modulation of nucleic acid structures, are key elements of the different mechanisms which make HNS such a versatile regulator.

A brief review of the molecular mechanisms of nickel carcinogenesis is presented. Molecular mechanisms of nickel carcinogenesis are considered from the pointofview of nickelinduced gene silencing by DNA hypermethylation in mammalian cells and by its ability to inhibit histone acetylation. Model systems designed to study the molecular mechanism of gene silencing are discussed.

We describe here our recent studies of the DNA binding properties of Msh2-Msh6 and Mlh1-Pms1, two protein complexes required to repair mismatches generated during DNA replication. Mismatched DNA binding by Msh2-Msh6 was probed by mutagenesis based on the crystal structure of the homologous bacterial MutS homodimer bound to DNA. The results suggest that several amino acid side chains inferred to interact with the DNA backbone near the mismatch are critical for repair activity. These contacts, which are different in Msh2 and Msh6, likely facilitate stacking and hydrogen bonding interactions between side chains in Msh6 and the mismatched base, thus stabilizing a kinked DNA conformation that permits subsequent repair steps coordinated by the Mlh1-Pms1 heterodimer. Mlh1-Pms1 also binds to DNA, but independently of a mismatch. Mlh1-Pms1 binds short DNA substrates with low affinity and with a slight preference for singlestranded DNA. It also binds longer duplex DNA molecules, but with a higher affinity indicative of cooperative binding. Indeed, imaging by atomic force microscopy reveals cooperative DNA binding and simultaneous interaction with two DNA duplexes. The novel DNA binding properties of Mlh1- Pms1 may be relevant to signal transduction during DNA mismatch repair and to recombination, meiosis and cellular responses to DNA damage.

In Salmonella typhimurium, a single enzyme catalyzes both the acetyl CoAdependent Oacetylation of hydroxylamines (a key step in the activation of mutagenic nitroaromatic compounds and related aromatic and heterocyclic amines) and the Nacetylation of aromatic amines. S. typhimurium Ames test mutants lacking this activity are highly resistant to the genotoxic effects of nitro compounds. However, such mutants have not yet been obtained in Escherichia coli. We used a PCRbased method to engineer a null mutation (deletion) of the nhoA gene encoding the enzyme in E. coli and we transduced this mutation into a lacZ strain background suitable for use in mutation assays. In E. coli, as in S. typhimurium, nhoA mutants show marked resistance to nitro compound mutagenicity. The new strains provide a clean background for expression of recombinant Nacetyltransferases.

Nacetyltransferase 2 (NAT2) catalyzes Nacetylation and Oacetylation of many drugs and environmental carcinogens. Genetic polymorphisms in the NAT2 gene have been associated with differential susceptibility to cancers and drug toxicity from these compounds. Single nucleotide polymorphisms (SNPs) have been identified in the human NAT2 coding region. A new allele, NAT2*19, possessing the C190T (R64W) exchange, was recently identified. In order to understand the effect of this new SNP, recombinant NAT2*4 (reference) and NAT2*19 were expressed in yeast (Schizosaccharomyces pombe). The C190T (R64W) SNP in NAT2*19 caused substantial reduction in the NAT2 protein level and stability, but did not cause significant reduction in transformation efficiency or mRNA level. The enzymatic activities for Nacetylation of two arylamine carcinogens (2-aminofluorene, 4-aminobiphenyl), and a sulfonamide drug (sulfamethazine) were over 100-fold lower for NAT2 19 compared to reference NAT2 4. Kinetic studies showed a reduction in Vmax but no significant change in substrate Km. In addition, the SNP caused significant reduction in the Oacetylation of the Nhydroxy 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine. These results show that NAT2*19 possessing the C190T (R64W) SNP encodes a slow acetylator phenotype for both N and Oacetylation, due to a reduction in the amount and stability of the NAT2 19 allozyme.

Ceramide has been suggested as an important mediator of apoptosis. In HT-29 colorectal cancer cells increased ceramide levels, induced by exogenous Nacetylsphingosine (NAS, also known as C2-ceramide) or by 1-phenyl-2-(decanoylamino)-3-morpholino-1- propanol (PDMP), inhibited the transport and processing of cathepsin D (CD), a lysosomal protease implicated in apoptosis of tumour cells. C2-dihydroceramide (DHC2), an inactive analogue of NAS, had no effect on CD transport and maturation. The treatment with either NAS or PDMP was revealed to be cytotoxic for HT-29 cells and led to cell death with classical features of apoptosis. Morphological signs of apoptosis and DNA fragmentation became apparent only between 24 and 48 h of incubation and poly(ADP ribose)polymerase cleavage, a hallmark of caspase 3 activity, occurred no earlier than 8 h from incubation. Secretion of proCD was almost abolished and the formation of doublechain mature CD was reduced and delayed by NAS, whereas PDMP largely inhibited the lysosomal targeting and maturation of proCD. NAS and PDMPinduced alteration of proCD transport and maturation were apparent already 2 h after incubation with the drugs, which is much earlier than when classical biochemical and morphological evidence of apoptosis could be detected. These data indicate that alteration of CD (and possibly of other glycoproteins) transport along the secretory pathway due to increased levels of cellassociated ceramide is an early event in cells undergoing apoptosis.

The apical multidrug resistance protein MRP2 (symbol ABCC2) is an ATPdependent export pump for anionic conjugates in polarized cells. MRP2 has only 48% amino acid identity with the paralog MRP1 (ABCC1). In this study we show that purified recombinant MRP2 reconstituted in proteoliposomes is functionally active in substrate transport. The Km values for ATP and LTC4 in the transport by MRP2 in proteoliposomes were 560 M and 450 nM, respectively. This transport function of MRP2 in proteoliposomes was dependent on the amount of MRP2 protein present and was determined to 2.7 pmol min exp.(-1) mg MRP2 exp.(-1) at 100 nM LTC[4]. Transport was competitively inhibited by the quinoline derivative MK571 with 50% inhibition at about 12 M. Our data document the first reconstitution of transportactive purified recombinant MRP2. Binding and immunoprecipitation experiments indicated that MRP2 preferentially associates with the chaperone calnexin, but coreconstitution studies using purified MRP2 and purified calnexin in proteoliposomes suggested that the LTC[4] transport function of MRP2 is not dependent on calnexin. The purified, transportactive MRP2 may serve to identify additional interacting proteins in the apical membrane of polarized cells.

The ectodomain of the human transferrin receptor (TfR) is released as soluble TfR into the blood by cleavage within a stalk. The major cleavage site is located Cterminally of Arg-100; alternative cleavage sites are also present. Since the cleavage process is still unclear, we looked for proteases involved in TfR ectodomain release. In the supernatant of U937 histiocytic cells we detected alternatively cleaved TfR (at Glu-110). In membrane fractions of these cells we identified two distinct proteolytic activities responsible for TfR cleavage within the stalk at either Val-108 or Lys-95. Both activities could be inhibited by serine protease inhibitors, but not by inhibitors of any other class of proteases. Protein purification yielded a 28 kDa protein that generated the Val-108 terminus. The protease activity could be ascribed to neutrophil elastase according to the substrate specificity determined by amino acid substitution analysis of synthetic peptides, an inhibitor profile, the size of the protease and the use of specific antibodies. The results of analogous experiments suggest that the second activity is represented by another serine protease, cathepsin G. Thus, membraneassociated forms of neutrophil elastase and cathepsin G may be involved in alternative TfR shedding in U937 cells.

Bovine spongiform encephalopathy has been epizootic in cows for the last two decades, and most probably causes variant CreutzfeldtJakob disease in humans. A thorough understanding of prion pathogenesis relies on suitable animal models. Modeling the transmission of BSE to primates is a crucial public health priority, necessary for determining the tissue distribution of the agent and for devising therapies. Susceptibility of humans to BSE is partly determined by polymorphism within the gene encoding the cellular prion protein, Prnp, a fact that must be taken into account in primate studies. However, no information is available on Prnp polymorphisms in primates. We have sequenced the Prnp open reading frames of 30 nonconsanguineous Rhesus macaques. All macaques were homozygous for methionine at codon 129, which is polymorphic in humans and seems to modulate prion susceptibility. However, we identified a novel polymorphism in macaque Prnp, localized on codon 226 (Y226F). A modulatory effect of this polymorphism on the development of prion disease is possible because codon 226 is close to the suggested binding side of the factor X, which has been invoked as a determinant of the prion species barrier.