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Where there's smoke... With the advent of large genomic data sets, geneticists can examine at a new level the influence of genes on behaviour. Two groups have conducted genome-wide association studies involving lung cancer, and both find that sequences in the nicotinic acetylcholine receptor subunit gene cluster contribute susceptibility, although the groups took different paths to this result. Hung et al . suggest that this susceptibility is not related to smoking status or frequency, and show association with a specific amino acid change. Thorgeirsson et al . find that alleles present in a cluster of nicotinic acid receptor genes do not influence whether or not a person smokes, but do affect the number of cigarettes smoked per day, and are therefore also associated with risk of lung cancer and peripheral arterial disease. Either way, the possible potential of nicotinic acetylcholine receptors as drug targets is underlined. A genome-wide association study for lung cancer finds that genetic sequences in the nicotinic acetylcholine receptor subunit gene cluster contribute susceptibility. Interestingly, this susceptibility is not related to smoking status or frequency, and seems to come from a change in an amino acid in the receptor itself. Lung cancer is the most common cause of cancer death worldwide, with over one million cases annually 1 . To identify genetic factors that modify disease risk, we conducted a genome-wide association study by analysing 317,139 single-nucleotide polymorphisms in 1,989 lung cancer cases and 2,625 controls from six central European countries. We identified a locus in chromosome region 15q25 that was strongly associated with lung cancer ( P = 9 × 10 -10 ). This locus was replicated in five separate lung cancer studies comprising an additional 2,513 lung cancer cases and 4,752 controls ( P = 5 × 10 -20 overall), and it was found to account for 14% (attributable risk) of lung cancer cases. Statistically similar risks were observed irrespective of smoking status or propensity to smoke tobacco. The association region contains several genes, including three that encode nicotinic acetylcholine receptor subunits ( CHRNA5 , CHRNA3 and CHRNB4 ). Such subunits are expressed in neurons and other tissues, in particular alveolar epithelial cells, pulmonary neuroendocrine cells and lung cancer cell lines 2 , 3 , and they bind to N ′-nitrosonornicotine and potential lung carcinogens 4 . A non-synonymous variant of CHRNA5 that induces an amino acid substitution (D398N) at a highly conserved site in the second intracellular loop of the protein is among the markers with the strongest disease associations. Our results provide compelling evidence of a locus at 15q25 predisposing to lung cancer, and reinforce interest in nicotinic acetylcholine receptors as potential disease candidates and chemopreventative targets 5 .

Mammals have nine different homologues (ALKBH1–9) of the Escherichia coli DNA repair demethylase AlkB. ALKBH2 is a genuine DNA repair enzyme, but the in vivo function of the other ALKBH proteins has remained elusive. It was recently shown that ALKBH8 contains an additional transfer RNA (tRNA) methyltransferase domain, which generates the wobble nucleoside 5-methoxycarbonylmethyluridine (mcm 5 U) from its precursor 5-carboxymethyluridine (cm 5 U). In this study, we report that ( R )- and ( S )-5-methoxycarbonylhydroxymethyluridine (mchm 5 U), hydroxylated forms of mcm 5 U, are present in mammalian , and , respectively, representing the first example of a diastereomeric pair of modified RNA nucleosides. Through in vitro and in vivo studies, we show that both diastereomers of mchm 5 U are generated from mcm 5 U, and that the AlkB domain of ALKBH8 specifically hydroxylates mcm 5 U into ( S )-mchm 5 U in . These findings expand the function of the ALKBH oxygenases beyond nucleic acid repair and increase the current knowledge on mammalian wobble uridine modifications and their biogenesis. Uridines at the wobble position of transfer RNA anticodons are usually modified to allow efficient decoding of messenger RNA codons. In this study, ALKBH8 is shown to be a bifunctional transfer RNA modification enzyme required for the formation of a novel diastereomeric pair of modified wobble uridines.

Both a DNA lesion and an intermediate for antibody maturation, uracil is primarily processed by base excision repair (BER), either initiated by uracil-DNA glycosylase (UNG) or by single-strand selective monofunctional uracil DNA glycosylase (SMUG1). The relative in vivo contributions of each glycosylase remain elusive. To assess the impact of SMUG1 deficiency, we measured uracil and 5-hydroxymethyluracil, another SMUG1 substrate, in Smug1 −/− mice. We found that 5-hydroxymethyluracil accumulated in Smug1 −/− tissues and correlated with 5-hydroxymethylcytosine levels. The highest increase was found in brain, which contained about 26-fold higher genomic 5-hydroxymethyluracil levels than the wild type. Smug1 −/− mice did not accumulate uracil in their genome and Ung −/− mice showed slightly elevated uracil levels. Contrastingly, Ung −/− Smug1 −/− mice showed a synergistic increase in uracil levels with up to 25-fold higher uracil levels than wild type. Whole genome sequencing of UNG/SMUG1-deficient tumours revealed that combined UNG and SMUG1 deficiency leads to the accumulation of mutations, primarily C to T transitions within CpG sequences. This unexpected sequence bias suggests that CpG dinucleotides are intrinsically more mutation prone. In conclusion, we showed that SMUG1 efficiently prevent genomic uracil accumulation, even in the presence of UNG, and identified mutational signatures associated with combined UNG and SMUG1 deficiency.

Uracil in DNA may result from incorporation of dUMP during replication and from spontaneous or enzymatic deamination of cytosine, resulting in U:A pairs or U:G mismatches, respectively. Uracil generated by activation-induced cytosine deaminase (AID) in B cells is a normal intermediate in adaptive immunity. Five mammalian uracil-DNA glycosylases have been identified; these are mitochondrial UNG1 and nuclear UNG2, both encoded by the UNG gene, and the nuclear proteins SMUG1, TDG and MBD4. Nuclear UNG2 is apparently the sole contributor to the post-replicative repair of U:A lesions and to the removal of uracil from U:G contexts in immunoglobulin genes as part of somatic hypermutation and class-switch recombination processes in adaptive immunity. All uracil-DNA glycosylases apparently contribute to U:G repair in other cells, but they are likely to have different relative significance in proliferating and non-proliferating cells, and in different phases of the cell cycle. There are also some indications that there may be species differences in the function of the uracil-DNA glycosylases.

Repair of DNA damage is essential for maintaining genome integrity, and repair deficiencies in mammals are associated with cancer, neurological disease and developmental defects 1 . Alkylation damage in DNA is repaired by at least three different mechanisms, including damage reversal by oxidative demethylation of 1-methyladenine and 3-methylcytosine by Escherichia coli AlkB 2 , 3 . By contrast, little is known about consequences and cellular handling of alkylation damage to RNA 4 . Here we show that two human AlkB homologues, hABH2 and hABH3, also are oxidative DNA demethylases and that AlkB and hABH3, but not hABH2, also repair RNA. Whereas AlkB and hABH3 prefer single-stranded nucleic acids, hABH2 acts more efficiently on double-stranded DNA. In addition, AlkB and hABH3 expressed in E. coli reactivate methylated RNA bacteriophage MS2 in vivo , illustrating the biological relevance of this repair activity and establishing RNA repair as a potentially important defence mechanism in living cells. The different catalytic properties and the different subnuclear localization patterns shown by the human homologues indicate that hABH2 and hABH3 have distinct roles in the cellular response to alkylation damage.

The ALKBH family of Fe(II) and 2-oxoglutarate dependent oxygenases comprises enzymes that display sequence homology to AlkB from E. coli, a DNA repair enzyme that uses an oxidative mechanism to dealkylate methyl and etheno adducts on the nucleobases. Humans have nine different ALKBH proteins, ALKBH1-8 and FTO. Mammalian and plant ALKBH8 are tRNA hydroxylases targeting 5-methoxycarbonylmethyl-modified uridine (mcm5U) at the wobble position of tRNAGly(UCC). In contrast, the genomes of some bacteria encode a protein with strong sequence homology to ALKBH8, and robust DNA repair activity was previously demonstrated for one such protein. To further explore this apparent functional duality of the ALKBH8 proteins, we have here enzymatically characterized a panel of such proteins, originating from bacteria, protozoa and mimivirus. All the enzymes showed DNA repair activity in vitro, but, interestingly, two protozoan ALKBH8s also catalyzed wobble uridine modification of tRNA, thus displaying a dual in vitro activity. Also, we found the modification status of tRNAGly(UCC) to be unaltered in an ALKBH8 deficient mutant of Agrobacterium tumefaciens, indicating that bacterial ALKBH8s have a function different from that of their eukaryotic counterparts. The present study provides new insights on the function and evolution of the ALKBH8 family of proteins.

Two human homologs of the Escherichia coli AlkB protein, denoted hABH2 and hABH3, were recently shown to directly reverse 1‐methyladenine (1meA) and 3‐methylcytosine (3meC) damages in DNA. We demonstrate that mice lacking functional mABH2 or mABH3 genes, or both, are viable and without overt phenotypes. Neither were histopathological changes observed in the gene‐targeted mice. However, in the absence of any exogenous exposure to methylating agents, mice lacking mABH2, but not mABH3 defective mice, accumulate significant levels of 1meA in the genome, suggesting the presence of a biologically relevant endogenous source of methylating agent. Furthermore, embryonal fibroblasts from mABH2‐deficient mice are unable to remove methyl methane sulfate (MMS)‐induced 1meA from genomic DNA and display increased cytotoxicity after MMS exposure. In agreement with these results, we found that in vitro repair of 1meA and 3meC in double‐stranded DNA by nuclear extracts depended primarily, if not solely, on mABH2. Our data suggest that mABH2 and mABH3 have different roles in the defense against alkylating agents.

Mice deficient in the Ung uracil-DNA glycosylase have an increased level of uracil in their genome, consistent with a major role of Ung counteracting U : A base pairs arising by misincorporation of dUMP during DNA replication. A complementary uracil-excising activity apparently acts on premutagenic U : G lesions resulting from deamination of cytosine throughout the genome. However, Ung specifically processes U : G lesions targeted to immunoglobulin variable (V) genes during somatic hypermutation and class-switch recombination. Gene-targeted Ung −/− null mice remained tumour-free and showed no overt pathological phenotype up to ∼12 months of age. We have monitored a large cohort of ageing Ung −/− mice and, beyond 18 months of age, they had a higher morbidity than Ung +/+ controls. Post-mortem analyses revealed pathological changes in lymphoid organs, abnormal lymphoproliferation, and a greatly increased incidence of B-cell lymphomas in older Ung-deficient mice. These are the first data reporting the development of spontaneous malignancies in mice due to deficiency in a DNA glycosylase. Furthermore, they support a specific role for Ung in the immune system, with lymphomagenesis being related to perturbed processing of antibody genes in germinal centre B cells.

ANY uracil bases in DNA, a result of either misincorporation or deamination of cytosine, are removed by uracil-DNA glycosylase (UDG), one of the most efficient and specific of the base-excision DNA-repair enzymes 1 . Crystal structures of human 2,3 and viral 4 UDGs complexed with free uracil have indicated that the enzyme binds an extrahelical uracil. Such binding of undamaged extrahelical bases has been seen in the structures of two bacterial methyltransferases 5,6 and bacteriophage T4 endonuclease V (ref. 7). Here we characterize the DNA binding and kinetics of several engineered human UDG mutants and present the crystal structure of one of these, which to our knowledge represents the first structure of any eukaryotic DNA repair enzyme in complex with its damaged, target DNA. Electrostatic orientation along the UDG active site, insertion of an amino acid (residue 272) into the DNA through the minor groove, and compression of the DNA backbone flanking the uracil all result in the flipping-out of the damaged base from the DNA major groove, allowing specific recognition of its phosphate, deoxyribose and uracil moieties. Our structure thus provides a view of a productive complex specific for cleavage of uracil from DNA and also reveals the basis for the enzyme-assisted nucleotide flipping by this critical DNA-repair enzyme.

Abasic [apurinic/apyrimidinic (AP)] sites are common, noncoding DNA lesions. Despite extensive investigation, the mutational pattern they provoke in eukaryotic cells remains unresolved. We constructed Saccharomyces cerevisiae strains in which chromosomal AP sites were generated during normal cell growth by altered human uracil-DNA glycosylases that remove undamaged cytosines or thymines. The mutation target was the URA3 gene inserted near the ARS309 origin to allow defined replication polarity. Expression of the altered glycosylases caused a 7- to 18-fold mutator effect in AP endonuclease-deficient ((delta)apn1) yeast, which depended highly on the known translesion synthesis enzymes Rev1 and DNA polymerase zota. For the C-glycosylase, GC>CG transversions were the predominant mutations, followed by GC>AT transitions. AT>CG transversions predominated for the T-glycosylase. These results support a major role for Rev1-dependent dCMP insertion across from AP sites and a lesser role for dAMP insertion. Unexpectedly, there was also a significant proportion of dTMP insertions that suggest another mutational pathway at AP sites. Although replication polarity did not strongly influence mutagenesis at AP sites, for certain mutation types, there was a surprisingly strong difference between the transcribed and non-transcribed strands of URA3. The basis for this strand discrimination requires further exploration.