NORMAL GENOMIC

• Chandola U, Das R, Panda B
• Role of the N6-methyladenosine RNA mark in gene regulation and its implications on development and disease.
• Brief Funct Genomics. 2015; 14: 169-79
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• Epigenetics is a field that encompasses chemical modifications of DNA and histone proteins, both of which alter gene expression without changing the underlying nucleotide sequence. DNA methylation and modifications of histone tails have been studied in detail and are now known to be global gene regulatory mechanisms. An analogous post-transcriptional modification is chemical modification of specific nucleotides in RNA. Study of RNA modifications is a nascent field as yet, and the significance of these marks in controlling cell growth and differentiation is just beginning to be appreciated. The addition of a methyl group to adenosine (N-methyl-6-adenosine) or m6A is the most abundant modification in mammalian mRNAs. Though identified four decades ago, interest in this particular modification was set off by the discovery that the obesity gene FTO was an RNA demethylase. Since then, many studies have investigated m6A modification in different species. In this review, we summarize the current literature and hypotheses about the presence and function of this ubiquitous RNA modification in mammals, viruses, yeast and plants in terms of the consensus sequence and the methyltransferase/demethylation machinery identified thus far. We discuss its potential role in regulating molecular and physiological processes in each of these organisms, especially its role in RNA splicing, RNA degradation and development. We also enlist the methodologies developed so far, both locus-specific and transcriptome-wide, to study this modification. Lastly, we discuss whether m6A alterations have consequences on modulating disease aetiology, and speculate about its potential role in cancer.

• Johansson C et al.
• The roles of Jumonji-type oxygenases in human disease.
• Epigenomics. 2014; 6: 89-120
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• The iron- and 2-oxoglutarate-dependent oxygenases constitute a phylogenetically conserved class of enzymes that catalyze hydroxylation reactions in humans by acting on various types of substrates, including metabolic intermediates, amino acid residues in different proteins and various types of nucleic acids. The discovery of jumonji (Jmj), the founding member of a class of Jmj-type chromatin modifying enzymes and transcriptional regulators, has culminated in the discovery of several branches of histone lysine demethylases, with essential functions in regulating the epigenetic landscape of the chromatin environment. This work has now been considerably expanded into other aspects of epigenetic biology and includes the discovery of enzymatic steps required for methyl-cytosine demethylation as well as modification of RNA and ribosomal proteins. This overview aims to summarize the current knowledge on the human Jmj-type enzymes and their involvement in human pathological processes, including development, cancer, inflammation and metabolic diseases.

• Dong C, Zhang H, Xu C, Arrowsmith CH, Min J
• Structure and function of dioxygenases in histone demethylation and DNA/RNA demethylation.
• IUCrJ. 2014; 1: 540-9
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• Iron(II) and 2-oxoglutarate (2OG)-dependent dioxygenases involved in histone and DNA/RNA demethylation convert the cosubstrate 2OG and oxygen to succinate and carbon dioxide, resulting in hydroxylation of the methyl group of the substrates and subsequent demethylation. Recent evidence has shown that these 2OG dioxygenases play vital roles in a variety of biological processes, including transcriptional regulation and gene expression. In this review, the structure and function of these dioxygenases in histone and nucleic acid demethylation will be discussed. Given the important roles of these 2OG dioxygenases, detailed analysis and comparison of the 2OG dioxygenases will guide the design of target-specific small-molecule chemical probes and inhibitors.

• Kuo PH, Chiang CH, Wang YT, Doudeva LG, Yuan HS
• The crystal structure of TDP-43 RRM1-DNA complex reveals the specific recognition for UG- and TG-rich nucleic acids.
• Nucleic Acids Res. 2014; 42: 4712-22
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• TDP-43 is an important pathological protein that aggregates in the diseased neuronal cells and is linked to various neurodegenerative disorders. In normal cells, TDP-43 is primarily an RNA-binding protein; however, how the dimeric TDP-43 binds RNA via its two RNA recognition motifs, RRM1 and RRM2, is not clear. Here we report the crystal structure of human TDP-43 RRM1 in complex with a single-stranded DNA showing that RRM1 binds the nucleic acid extensively not only by the conserved beta-sheet residues but also by the loop residues. Mutational and biochemical assays further reveal that both RRMs in TDP-43 dimers participate in binding of UG-rich RNA or TG-rich DNA with RRM1 playing a dominant role and RRM2 playing a supporting role. Moreover, RRM1 of the amyotrophic lateral sclerosis-linked mutant D169G binds DNA as efficiently as the wild type; nevertheless, it is more resistant to thermal denaturation, suggesting that the resistance to degradation is likely linked to TDP-43 proteinopathies. Taken together all the data, we suggest a model showing that the two RRMs in each protomer of TDP-43 homodimer work together in RNA binding and thus the dimeric TDP-43 recognizes long clusters of UG-rich RNA to achieve high affinity and specificity.

• Zheng G et al.
• Synthesis of a FTO inhibitor with anticonvulsant activity.
• ACS Chem Neurosci. 2014; 5: 658-65
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• We describe the rationale for and the synthesis of a new class of compounds utilizing a modular approach that are designed to mimic ascorbic acid and to inhibit 2-oxoglutarate-dependent hydroxylases. Preliminary characterization of one of these compounds indicates in vivo anticonvulsant activity (6 Hz mouse model) at nontoxic doses, inhibition of the 2-oxoglutarate-dependent hydroxylase FTO, and expected increase in cellular N(6)-methyladenosine. This compound is also able to modulate various microRNA, an interesting result in light of the recent view that modulation of microRNAs may be useful for the treatment of CNS disease.

• Muller TD, Tschop MH, Hofmann S
• Emerging function of fat mass and obesity-associated protein (fto).
• PLoS Genet. 2013; 9: 1003223-1003223

• Zielke LG, Bortfeldt RH, Reissmann M, Tetens J, Thaller G, Brockmann GA
• Impact of variation at the FTO locus on milk fat yield in Holstein dairy cattle.
• PLoS One. 2013; 8: 63406-63406
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• This study explores the biological role of the Fat Mass and Obesity associated (FTO) gene locus on milk composition in German Holstein cattle. Since FTO controls energy homeostasis and expenditure and the FTO locus has repeatedly shown association with obesity in human studies, we tested FTO as a candidate gene in particular for milk fat yield, which represents a high amount of energy secreted during lactation. The study was performed on 2,402 bulls and 860 cows where dense milk composition data were available. Genetic information was taken from a 2 Mb region around FTO. Five SNPs and two haplotype blocks in a 725 kb region covering FTO and the neighboring genes RPGRIP1L, U6ATAC, and 5 S rRNA were associated with milk fat yield and also affected protein yield in the same direction. Interestingly, higher frequency SNP alleles and haplotypes within the FTO gene increased milk fat and protein yields by up to 2.8 and 2.2 kg per lactation, respectively, while the most frequent haplotype in the upstream block covering exon 1 of FTO to exon 15 of RPGRIP1L had opposite effects with lower fat and milk yield. Both haplotype blocks were also significant in cows. The loci accounted for about 1% of the corresponding trait variance in the population. The association signals not only provided evidence for at least two causative mutations in the FTO locus with a functional effect on milk but also milk protein yield. The pleiotropic effects suggest a biological function on the usage of energy resources and the control of energy balance rather than directly affecting fat and protein synthesis. The identified effect of the obesity gene locus on milk energy content suggests an impact on infant nutrition by breast feeding in humans.

• da Silva CF et al.
• Association between a frequent variant of the FTO gene and anthropometric phenotypes in Brazilian children.
• BMC Med Genet. 2013; 14: 34-34
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• BACKGROUND: Our goal was to analyze the association of the fat mass and obesity- associated (FTO) gene rs9939609 variant (T/A) with the anthropometric and dietary intake phenotypes related to obesity in Brazilian children. METHODS: We analyzed the association of this single nucleotide polymorphism (SNP) with phenotypes related to the accumulation of body mass in a cohort of 348 children followed from the time of birth until 8 years old and then replicated the main findings in an independent schoolchildren sample (n = 615). RESULTS: At the age of 4, we observed a significant association between the A/A genotype and a higher mean BMI Z-score (P = 0.036). At the age of 8, the A/A individuals still presented with a higher BMI Z-score (P = 0.011) and with marginal differences in the volume of subcutaneous fat (P = 0.048). We replicated these findings in the schoolchildren sample, which showed that those with at least one copy of the A allele presented with a higher BMI Z-score (P = 0.029) and volume of subcutaneous fat (P = 0.016). CONCLUSION: Our results indicate that this FTO variant is associated with increased body mass and subcutaneous fat in Brazilian children beginning at the age of 4.

• Zhou B, Han Z
• Crystallization and preliminary X-ray diffraction of the RNA demethylase ALKBH5.
• Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013; 69: 1231-4
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• N(6)-methyladenosine (m6A) is a ubiquitous modification found in mammalian mRNA and long noncoding RNA. ALKBH5 is a member of the iron(II)- and 2-oxoglutarate-dependent AlkB oxygenase family and has been shown to catalyze the oxidative demethylation of N(6)-methyladenosine in RNA. The ALKBH5 protein was purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 A resolution using synchrotron radiation. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 57.456, b = 83.406, c = 92.909 A, alpha = beta = gamma = 90.00 degrees and one molecule in the asymmetric unit.

• Melka MG et al.
• FTO, obesity and the adolescent brain.
• Hum Mol Genet. 2013; 22: 1050-8
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• Genetic variations in fat mass- and obesity (FTO)-associated gene, a well-replicated gene locus of obesity, appear to be associated also with reduced regional brain volumes in elderly. Here, we examined whether FTO is associated with total brain volume in adolescence, thus exploring possible developmental effects of FTO. We studied a population-based sample of 598 adolescents recruited from the French Canadian founder population in whom we measured brain volume by magnetic resonance imaging. Total fat mass was assessed with bioimpedance and body mass index was determined with anthropometry. Genotype-phenotype associations were tested with Merlin under an additive model. We found that the G allele of FTO (rs9930333) was associated with higher total body fat [TBF (P = 0.002) and lower brain volume (P = 0.005)]. The same allele was also associated with higher lean body mass (P = 0.03) and no difference in height (P = 0.99). Principal component analysis identified a shared inverse variance between the brain volume and TBF, which was associated with FTO at P = 5.5 x 10(-6). These results were replicated in two independent samples of 413 and 718 adolescents, and in a meta-analysis of all three samples (n = 1729 adolescents), FTO was associated with this shared inverse variance at P = 1.3 x 10(-9). Co-expression networks analysis supported the possibility that the underlying FTO effects may occur during embryogenesis. In conclusion, FTO is associated with shared inverse variance between body adiposity and brain volume, suggesting that this gene may exert inverse effects on adipose and brain tissues. Given the completion of the overall brain growth in early childhood, these effects may have their origins during early development.

• Yi C, He C
• DNA repair by reversal of DNA damage.
• Cold Spring Harb Perspect Biol. 2013; 5: 12575-12575
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• Endogenous and exogenous factors constantly challenge cellular DNA, generating cytotoxic and/or mutagenic DNA adducts. As a result, organisms have evolved different mechanisms to defend against the deleterious effects of DNA damage. Among these diverse repair pathways, direct DNA-repair systems provide cells with simple yet efficient solutions to reverse covalent DNA adducts. In this review, we focus on recent advances in the field of direct DNA repair, namely, photolyase-, alkyltransferase-, and dioxygenase-mediated repair processes. We present specific examples to describe new findings of known enzymes and appealing discoveries of new proteins. At the end of this article, we also briefly discuss the influence of direct DNA repair on other fields of biology and its implication on the discovery of new biology.

• Fu Y et al.
• FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA.
• Nat Commun. 2013; 4: 1798-1798
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• N(6)-methyladenosine is a prevalent internal modification in messenger RNA and non-coding RNA affecting various cellular pathways. Here we report the discovery of two additional modifications, N(6)-hydroxymethyladenosine (hm(6)A) and N(6)-formyladenosine (f(6)A), in mammalian messenger RNA. We show that Fe(II)- and alpha-ketoglutarate-dependent fat mass and obesity-associated (FTO) protein oxidize N(6)-methyladenosine to generate N(6)-hydroxymethyladenosine as an intermediate modification, and N(6)-formyladenosine as a further oxidized product. N(6)-hydroxymethyladenosine and N(6)-formyladenosine have half-life times of ~3 h in aqueous solution under physiological relevant conditions, and are present in isolated messenger RNA from human cells as well as mouse tissues. These previously unknown modifications derived from the prevalent N(6)-methyladenosine in messenger RNA, formed through oxidative RNA demethylation, may dynamically modulate RNA-protein interactions to affect gene expression regulation.

• Simmons JM, Koslowsky DJ, Hausinger RP
• Characterization of a Trypanosoma brucei Alkb homolog capable of repairing alkylated DNA.
• Exp Parasitol. 2012; 131: 92-100
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• Trypanosoma brucei encodes a protein (denoted TbABH) that is homologous to AlkB of Escherichia coli and AlkB homolog (ABH) proteins in other organisms, raising the possibility that trypanosomes catalyze oxidative repair of alkylation-damaged DNA. TbABH was cloned and expressed in E. coli, and the recombinant protein was purified and characterized. Incubation of anaerobic TbABH with Fe(II) and alpha-ketoglutarate (alphaKG) produces a characteristic metal-to-ligand charge-transfer chromophore, confirming its membership in the Fe(II)/alphaKG dioxygenase superfamily. The protein binds to DNA, with a clear preference for alkylated oligonucleotides according to results derived by electrophoretic mobility shift assays. Finally, the protozoan gene was shown to partially complement E. coli alkB cells when stressed with methylmethanesulfonate; thus confirming assignment of TbABH as a functional AlkB protein in T. brucei.

• Tiwari HK et al.
• Association of Allelic Variation in Genes Mediating Aspects of Energy Homeostasis with Weight Gain during Administration of Antipsychotic Drugs (CATIE Study).
• Front Genet. 2011; 2: 56-56
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• Antipsychotic drugs are widely used in treating schizophrenia, bipolar disorder, and other psychiatric disorders. Many of these drugs, despite their therapeutic advantages, substantially increase body weight. We assessed the association of alleles of 31 genes implicated in body weight regulation with weight gain among patients being treated with specific antipsychotic medications in the clinical antipsychotic trials in intervention effectiveness study, we found that rs2237988 in Potassium Channel Inwardly Rectifying Subfamily J Member 11 (KCNJ11), rs13269119 in Solute carrier family 30 member 8 (SLC30A8), and rs9922047 in fat mass and obesity associated (FTO) were associated with percent weight gain. We also observed the significant interaction of rs11643744 by treatment effect on the weight gain.

• Cheung MK, Yeo GS
• FTO Biology and Obesity: Why Do a Billion of Us Weigh 3 kg More?
• Front Endocrinol (Lausanne). 2011; 2: 4-4
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• Few would dispute that the current obesity epidemic has been driven by lifestyle and environmental changes. However, it is clear that individuals respond differently to these "obesigenic" changes and this variation in response has a strong genetic element. Genome-wide association studies have revealed that single nucleotide polymorphisms in Fat mass and obesity-associated transcript (FTO) are robustly associated with body mass index and obesity. Although the effect of these risk alleles are modest, with heterozygous and homozygous carriers weighing approximately 1.5 and 3 kg more respectively, there are an estimated one billion homozygous carriers in the world, spanning multiple different ethnicities and populations. Yet despite its broad impact, the biological function of FTO, particularly its role in controlling energy balance, remains unknown. Although the study of severe Mendelian obesity has been invaluable in illuminating critical pathways controlling food intake, the major burden of disease is carried by those of us with "common obesity," which to date has resisted yielding meaningful biological insights. FTO has at last given us a handle on a huge, worldwide, common problem. In this review, we focus on the available genetic and in vivo evidence to date that implicates FTO in the control of energy balance.

• Choquet H, Meyre D
• Genetics of Obesity: What have we Learned?
• Curr Genomics. 2011; 12: 169-79
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• Candidate gene and genome-wide association studies have led to the discovery of nine loci involved in Mendelian forms of obesity and 58 loci contributing to polygenic obesity. These loci explain a small fraction of the heritability for obesity and many genes remain to be discovered. However, efforts in obesity gene identification greatly modified our understanding of this disorder. In this review, we propose an overlook of major lessons learned from 15 years of research in the field of genetics and obesity. We comment on the existence of the genetic continuum between monogenic and polygenic forms of obesity that pinpoints the role of genes involved in the central regulation of food intake and genetic predisposition to obesity. We explain how the identification of novel obesity predisposing genes has clarified unsuspected biological pathways involved in the control of energy balance that have helped to understand past human history and to explore causality in epidemiology. We provide evidence that obesity predisposing genes interact with the environment and influence the response to treatment relevant to disease prediction.

• Bjornstad LG, Zoppellaro G, Tomter AB, Falnes PO, Andersson KK
• Spectroscopic and magnetic studies of wild-type and mutant forms of the Fe(II)- and 2-oxoglutarate-dependent decarboxylase ALKBH4.
• Biochem J. 2011; 434: 391-8
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• The Fe(II)/2OG (2-oxoglutarate)-dependent dioxygenase superfamily comprises proteins that couple substrate oxidation to decarboxylation of 2OG to succinate. A member of this class of mononuclear non-haem Fe proteins is the Escherichia coli DNA/RNA repair enzyme AlkB. In the present work, we describe the magnetic and optical properties of the yet uncharacterized human ALKBH4 (AlkB homologue). Through EPR and UV-visible spectroscopy studies, we address the Fe-binding environment of the proposed catalytic centre of wild-type ALKBH4 and an Fe(II)-binding mutant. We could observe a novel unusual Fe(III) high-spin EPR-active species in the presence of sulfide with a g(max) of 8.2. The Fe(II) site was probed with NO. An intact histidine-carboxylate site is necessary for productive Fe binding. We also report the presence of a unique cysteine-rich motif conserved in the N-terminus of ALKBH4 orthologues, and investigate its possible Fe-binding ability. Furthermore, we show that recombinant ALKBH4 mediates decarboxylation of 2OG in absence of primary substrate. This activity is dependent on Fe as well as on residues predicted to be involved in Fe(II) co-ordination. The present results demonstrate that ALKBH4 represents an active Fe(II)/2OG-dependent decarboxylase and suggest that the cysteine cluster is involved in processes other than Fe co-ordination.

• Tan S, Nagai K
• Diversity makes for a rich tapestry.
• Curr Opin Struct Biol. 2011; 21: 81-2

• Wang H et al.
• Crystal structure of the human CNOT6L nuclease domain reveals strict poly(A) substrate specificity.
• EMBO J. 2010; 29: 2566-76
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• CCR4, an evolutionarily conserved member of the CCR4-NOT complex, is the main cytoplasmic deadenylase. It contains a C-terminal nuclease domain with homology to the endonuclease-exonuclease-phosphatase (EEP) family of enzymes. We have determined the high-resolution three-dimensional structure of the nuclease domain of CNOT6L, a human homologue of CCR4, by X-ray crystallography using the single-wavelength anomalous dispersion method. This first structure of a deadenylase belonging to the EEP family adopts a complete alpha/beta sandwich fold typical of hydrolases with highly conserved active site residues similar to APE1. The active site of CNOT6L should recognize the RNA substrate through its negatively charged surface. In vitro deadenylase assays confirm the critical active site residues and show that the nuclease domain of CNOT6L exhibits full Mg(2+)-dependent deadenylase activity with strict poly(A) RNA substrate specificity. To understand the structural basis for poly(A) RNA substrate binding, crystal structures of the CNOT6L nuclease domain have also been determined in complex with AMP and poly(A) DNA. The resulting structures suggest a molecular deadenylase mechanism involving a pentacovalent phosphate transition.

• Takao M et al.
• Human Nei-like protein NEIL3 has AP lyase activity specific for single-stranded DNA and confers oxidative stress resistance in Escherichia coli mutant.
• Genes Cells. 2009; 14: 261-70
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• Oxidative base damage leads to alteration of genomic information and is implicated as a cause of aging and carcinogenesis. To combat oxidative damage to DNA, cells contain several DNA glycosylases including OGG1, NTH1 and the Nei-like proteins, NEIL1 and NEIL2. A third Nei-like protein, NEIL3, is composed of an amino-terminal Nei-like domain and an unknown carboxy-terminal domain. In contrast to the other well-described DNA glycosylases, the DNA glycosylase activity and in vivo repair function of NEIL3 remains unclear. We show here that the structural modeling of the putative NEIL3 glycosylase domain (1-290) fits well to the known Escherichia coli Fpg crystal structure. In spite of the structural similarity, the recombinant NEIL3 and NEIL3(1-290) proteins do not cleave any of several test oligonucleotides containing a single modified base. Within the substrates, we detected AP lyase activity for single-stranded (ss) DNA but double-stranded (ds) DNA. The activity is abrogated completely in mutants with an amino-terminal deletion and at the zinc-finger motif. Surprisingly, NEIL3 partially rescues an E. coli nth nei mutant from hydrogen peroxide sensitivity. Taken together, repair of certain base damage including base loss in ssDNA may be mediated by NEIL3.

• Yi C, Yang CG, He C
• A non-heme iron-mediated chemical demethylation in DNA and RNA.
• Acc Chem Res. 2009; 42: 519-29
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• DNA methylation is arguably one of the most important chemical signals in biology. However, aberrant DNA methylation can lead to cytotoxic or mutagenic consequences. A DNA repair protein in Escherichia coli, AlkB, corrects some of the unwanted methylations of DNA bases by a unique oxidative demethylation in which the methyl carbon is liberated as formaldehyde. The enzyme also repairs exocyclic DNA lesions--that is, derivatives in which the base is augmented with an additional heterocyclic subunit--by a similar mechanism. Two proteins in humans that are homologous to AlkB, ABH2 and ABH3, repair the same spectrum of lesions; another human homologue of AlkB, FTO, is linked to obesity. In this Account, we describe our studies of AlkB, ABH2, and ABH3, including our development of a general strategy to trap homogeneous protein-DNA complexes through active-site disulfide cross-linking. AlkB uses a non-heme mononuclear iron(II) and the cofactors 2-ketoglutarate (2KG) and dioxygen to effect oxidative demethylation of the DNA base lesions 1-methyladenine (1-meA), 3-methylcytosine (3-meC), 1-methylguanine (1-meG), and 3-methylthymine (3-meT). ABH3, like AlkB, works better on single-stranded DNA (ssDNA) and is capable of repairing damaged bases in RNA. Conversely, ABH2 primarily repairs lesions in double-stranded DNA (dsDNA); it is the main housekeeping enzyme that protects the mammalian genome from 1-meA base damage. The AlkB-family proteins have moderate affinities for their substrates and bind DNA in a non-sequence-specific manner. Knowing that these proteins flip the damaged base out from the duplex DNA and insert it into the active site for further processing, we first engineered a disulfide cross-link in the active site to stabilize the Michaelis complex. Based on the detailed structural information afforded by the active-site cross-linked structures, we can readily install a cross-link away from the active site to obtain the native-like structures of these complexes. The crystal structures show a distinct base-flipping feature in AlkB and establish ABH2 as a dsDNA repair protein. They also provide a molecular framework for understanding the demethylation reaction catalyzed by these proteins and help to explain their substrate preferences. The chemical cross-linking method demonstrated here can be applied to trap other labile protein-DNA interactions and can serve as a general strategy for exploring the structural and functional aspects of base-flipping proteins.

• Timpson NJ et al.
• The fat mass- and obesity-associated locus and dietary intake in children.
• Am J Clin Nutr. 2008; 88: 971-8
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• BACKGROUND: A region of chromosome 16 containing the fat mass-and obesity-associated gene (FTO) is reproducibly associated with fat mass and body mass index (BMI), risk of obesity, and adiposity. OBJECTIVES: We aimed to assess the possibility that appetite plays a role in the association between FTO and BMI. DESIGN: Detailed dietary report information from the Avon Longitudinal Study of Parents and Children allowed the exploration of relations between FTO variation and dietary intake. Analyses were performed to investigate possible associations between variation at the FTO locus and the intake of a range of micronutrients and macronutrients, with adjustment for the bias often found within dietary report data when factors related to BMI are assessed. To test the hypothesis that FTO may be influencing appetite directly, rather than indirectly via BMI and altered intake requirements, we also assessed associations between FTO and dietary intake independent of BMI. RESULTS: Relations between a single-nucleotide polymorphism characterizing the FTO signal (rs9939609) and dietary variables were found and can be summarized by the effect of each additional allele (per-allele effects) on total energy and total fat (P < 0.001 for both). These associations were attenuated, but they persisted specifically for fat and energy consumption after adjustment for BMI [total daily fat consumption: approximately 1.5 g/d (P = 0.02 for the per-allele difference); total daily energy consumption: approximately 25 kJ/d (P = 0.03 for the per-allele difference)]. CONCLUSION: These associations suggest that persons carrying minor variants at rs9939609 were consuming more fat and total energy than were those not carrying such variants. They also suggest that this difference was not simply dependent on having higher average BMIs among the former group.

• Shioi S et al.
• Crystal structure of a biologically functional form of PriB from Escherichia coli reveals a potential single-stranded DNA-binding site.
• Biochem Biophys Res Commun. 2005; 326: 766-76
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• PriB is not only an essential protein necessary for the replication restart on the collapsed and disintegrated replication fork, but also an important protein for assembling of primosome onto PhiX174 genomic DNA during replication initiation. Here we report a 2.0-A-resolution X-ray structure of a biologically functional form of PriB from Escherichia coli. The crystal structure revealed that despite a low level of primary sequence identity, the PriB monomer, as well as the dimeric form, are structurally identical to the N-terminal DNA-binding domain of the single-stranded DNA-binding protein (SSB) from Escherichia coli, which possesses an oligonucleotides-binding-fold. The oligonucleotide-PriB complex model based on the oligonucleotides-SSB complex structure suggested that PriB had a DNA-binding pocket conserved in SSB from Escherichia coli and might bind to single-stranded DNA in the manner of SSB. Furthermore, surface plasmon resonance analysis and fluorescence measurements demonstrated that PriB binds single-stranded DNA with high affinity, by involving tryptophan residue. The significance of these results with respect to the functional role of PriB in the assembly of primosome is discussed.

• Han S, Arvai AS, Clancy SB, Tainer JA
• Crystal structure and novel recognition motif of rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis.
• J Mol Biol. 2001; 305: 95-107
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• Clostridium botulinum C3 exoenzyme inactivates the small GTP-binding protein family Rho by ADP-ribosylating asparagine 41, which depolymerizes the actin cytoskeleton. C3 thus represents a major family of the bacterial toxins that transfer the ADP-ribose moiety of NAD to specific amino acids in acceptor proteins to modify key biological activities in eukaryotic cells, including protein synthesis, differentiation, transformation, and intracellular signaling. The 1.7 A resolution C3 exoenzyme structure establishes the conserved features of the core NAD-binding beta-sandwich fold with other ADP-ribosylating toxins despite little sequence conservation. Importantly, the central core of the C3 exoenzyme structure is distinguished by the absence of an active site loop observed in many other ADP-ribosylating toxins. Unlike the ADP-ribosylating toxins that possess the active site loop near the central core, the C3 exoenzyme replaces the active site loop with an alpha-helix, alpha3. Moreover, structural and sequence similarities with the catalytic domain of vegetative insecticidal protein 2 (VIP2), an actin ADP-ribosyltransferase, unexpectedly implicates two adjacent, protruding turns, which join beta5 and beta6 of the toxin core fold, as a novel recognition specificity motif for this newly defined toxin family. Turn 1 evidently positions the solvent-exposed, aromatic side-chain of Phe209 to interact with the hydrophobic region of Rho adjacent to its GTP-binding site. Turn 2 evidently both places the Gln212 side-chain for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD ribose and holds the key Glu214 catalytic side-chain in the adjacent catalytic pocket. This proposed bipartite ADP-ribosylating toxin turn-turn (ARTT) motif places the VIP2 and C3 toxin classes into a single ARTT family characterized by analogous target protein recognition via turn 1 aromatic and turn 2 hydrogen-bonding side-chain moieties. Turn 2 centrally anchors the catalytic Glu214 within the ARTT motif, and furthermore distinguishes the C3 toxin class by a conserved turn 2 Gln and the VIP2 binary toxin class by a conserved turn 2 Glu for appropriate target side-chain hydrogen-bonding recognition. Taken together, these structural results provide a molecular basis for understanding the coupled activity and recognition specificity for C3 and for the newly defined ARTT toxin family, which acts in the depolymerization of the actin cytoskeleton. This beta5 to beta6 region of the toxin fold represents an experimentally testable and potentially general recognition motif region for other ADP-ribosylating toxins that have a similar beta-structure framework.

• Chapados BR, Chai Q, Hosfield DJ, Qiu J, Shen B, Tainer JA
• Structural biochemistry of a type 2 RNase H: RNA primer recognition and removal during DNA replication.
• J Mol Biol. 2001; 307: 541-56
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• DNA replication and cellular survival requires efficient removal of RNA primers during lagging strand DNA synthesis. In eukaryotes, RNA primer removal is initiated by type 2 RNase H, which specifically cleaves the RNA portion of an RNA-DNA/DNA hybrid duplex. This conserved type 2 RNase H family of replicative enzymes shares little sequence similarity with the well-characterized prokaryotic type 1 RNase H enzymes, yet both possess similar enzymatic properties. Crystal structures and structure-based mutational analysis of RNase HII from Archaeoglobus fulgidus, both with and without a bound metal ion, identify the active site for type 2 RNase H enzymes that provides the general nuclease activity necessary for catalysis. The two-domain architecture of type 2 RNase H creates a positively charged binding groove and links the unique C-terminal helix-loop-helix cap domain to the active site catalytic domain. This architectural arrangement apparently couples directional A-form duplex binding, by a hydrogen-bonding Arg-Lys phosphate ruler motif, to substrate-discrimination, by a tyrosine finger motif, thereby providing substrate-specific catalytic activity. Combined kinetic and mutational analyses of structurally implicated substrate binding residues validate this binding mode. These structural and mutational results together suggest a molecular mechanism for type 2 RNase H enzymes for the specific recognition and cleavage of RNA in the RNA-DNA junction within hybrid duplexes, which reconciles the broad substrate binding affinity with the catalytic specificity observed in biochemical assays. In combination with a recent independent structural analysis, these results furthermore identify testable molecular hypotheses for the activity and function of the type 2 RNase H family of enzymes, including structural complementarity, substrate-mediated conformational changes and coordination with subsequent FEN-1 activity.

• Morikawa K, Shirakawa M
• Three-dimensional structural views of damaged-DNA recognition: T4 endonuclease V, E. coli Vsr protein, and human nucleotide excision repair factor XPA.
• Mutat Res. 2000; 460: 257-75
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• Genetic information is frequently disturbed by introduction of modified or mismatch bases into duplex DNA, and hence all organisms contain DNA repair systems to restore normal genetic information by removing such damaged bases or nucleotides and replacing them by correct ones. The understanding of this repair mechanism is a central subject in cell biology. This review focuses on the three-dimensional structural views of damaged DNA recognition by three proteins. The first protein is T4 endonuclease V (T4 endo V), which catalyzes the first reaction step of the excision repair pathway to remove pyrimidine-dimers (PD) produced within duplex DNA by UV irradiation. The crystal structure of this enzyme complexed with DNA containing a thymidine-dimer provided the first direct view of DNA lesion recognition by a repair enzyme, indicating that the DNA kink coupled with base flipping-out is important for damaged DNA recognition. The second is very short patch repair (Vsr) endonuclease, which recognizes a TG mismatch within the five base pair consensus sequence. The crystal structure of this enzyme in complex with duplex DNA containing a TG mismatch revealed a novel mismatch base pair recognition scheme, where three aromatic residues intercalate from the major groove into the DNA to strikingly deform the base pair stacking but the base flipping-out does not occur. The third is human nucleotide excision repair (NER) factor XPA, which is a major component of a large protein complex. This protein has been shown to bind preferentially to UV- or chemical carcinogen-damaged DNA. The solution structure of the XPA central domain, essential for the interaction of damaged DNA, was determined by NMR. This domain was found to be divided mainly into a (Cys)4-type zinc-finger motif subdomain for replication protein A (RPA) recognition and the carboxyl terminal subdomain responsible for DNA binding.

• Pyle AM, Chu VT, Jankowsky E, Boudvillain M
• Using DNAzymes to cut, process, and map RNA molecules for structural studies or modification.
• Methods Enzymol. 2000; 317: 140-6

• Brautigam CA, Steitz TA
• Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes.
• Curr Opin Struct Biol. 1998; 8: 54-63
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• New levels in the understanding of DNA replication have been achieved from recent crystal structure determinations of several DNA polymerases and their substrate complexes. The structure of an alpha family DNA polymerase from bacteriophage RB69 shows some similarities, but also considerable differences in structure and organization from the pol I family DNA polymerases. Also, the functions of three polymerase domains and their conserved residues have been clarified by studying structures of pol I family DNA polymerases complexed to their substrates. These structures also confirm that an identical two-metal ion catalytic mechanism proposed previously is used by both the nonhomologous pol I and pol beta family DNA polymerases.

• Sclavi B, Woodson S, Sullivan M, Chance MR, Brenowitz M
• Time-resolved synchrotron X-ray "footprinting", a new approach to the study of nucleic acid structure and function: application to protein-DNA interactions and RNA folding.
• J Mol Biol. 1997; 266: 144-59
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• Hydroxyl radicals (.OH) can cleave the phosphodiester backbone of nucleic acids and are valuable reagents in the study of nucleic acid structure and protein-nucleic acid interactions. Irradiation of solutions by high flux "white light" X-ray beams based on bending magnet beamlines at the National Synchrotron Light Source (NSLS) yields sufficient concentrations of .OH so that quantitative nuclease protection ("footprinting") studies of DNA and RNA can be conducted with a duration of exposure in the range of 50 to 100 ms. The sensitivity of DNA and RNA to X-ray mediated .OH cleavage is equivalent. Both nucleic acids are completely protected from synchrotron X-ray induced cleavage by the presence of thiourea in the sample solution, demonstrating that cleavage is suppressed by a free radical scavenger. The utility of this time-dependent approach to footprinting is demonstrated with a synchrotron X-ray footprint of a protein-DNA complex and by a time-resolved footprinting analysis of the Mg(2+)-dependent folding of the Tetrahymena thermophilia L-21 ScaI ribozyme RNA. Equilibrium titrations reveal differences among the ribozyme domains in the cooperativity of Mg(2+)-dependent .OH protection. RNA .OH protection progress curves were obtained for several regions of the ribozyme over timescales of 30 seconds to several minutes. Progress curves ranging from > or = 3.5 to 0.4 min-1 were obtained for the P4-P6 and P5 sub-domains and the P3-P7 domain, respectively. The .OH protection progress curves have been correlated with the available biochemical, structural and modeling data to generate a model of the ribozyme folding pathway. Rate differences observed for specific regions within domains provide evidence for steps in the folding pathway not previously observed. Synchrotron X-ray footprinting is a new approach of general applicability for the study of time-resolved structural changes of nucleic acid conformation and protein-nucleic acid complexes.

• Mol CD et al.
• Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis.
• Cell. 1995; 80: 869-78
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• Crystal structures of the DNA repair enzyme human uracil-DNA glycosylase (UDG), combined with mutational analysis, reveal the structural basis for the specificity of the enzyme. Within the classic alpha/beta fold of UDG, sequence-conserved residues form a positively charged, active-site groove the width of duplex DNA, at the C-terminal edge of the central four-stranded parallel beta sheet. In the UDG-6-aminouracil complex, uracil binds at the base of the groove within a rigid preformed pocket that confers selectivity for uracil over other bases by shape complementary and by main chain and Asn-204 side chain hydrogen bonds. Main chain nitrogen atoms are positioned to stabilize the oxyanion intermediate generated by His-268 acting via nucleophilic attack or general base mechanisms. Specific binding of uracil flipped out from a DNA duplex provides a structural mechanism for damaged base recognition.

• Tucker PA, Tsernoglou D, Tucker AD, Coenjaerts FE, Leenders H, van der Vliet PC
• Crystal structure of the adenovirus DNA binding protein reveals a hook-on model for cooperative DNA binding.
• EMBO J. 1994; 13: 2994-3002
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• The adenovirus single-stranded DNA binding protein (Ad DBP) is a multifunctional protein required, amongst other things, for DNA replication and transcription control. It binds to single- and double-stranded DNA, as well as to RNA, in a sequence-independent manner. Like other single-stranded DNA binding proteins, it binds ssDNA, cooperatively. We report the crystal structure, at 2.6 A resolution, of the nucleic acid binding domain. This domain is active in DNA replication. The protein contains two zinc atoms in different, novel coordinations. The zinc atoms appear to be required for the stability of the protein fold rather than being involved in direct contacts with the DNA. The crystal structure shows that the protein contains a 17 amino acid C-terminal extension which hooks onto a second molecule, thereby forming a protein chain. Deletion of this C-terminal arm reduces cooperativity in DNA binding, suggesting a hook-on model for cooperativity. Based on this structural work and mutant studies, we propose that DBP forms a protein core around which the single-stranded DNA winds.

• Traub P, Mothes E, Shoeman R, Kuhn S, Scherbarth A
• Characterization of the nucleic acid-binding activities of the isolated amino-terminal head domain of the intermediate filament protein vimentin reveals its close relationship to the DNA-binding regions of some prokaryotic single-stranded DNA-binding proteins.
• J Mol Biol. 1992; 228: 41-57
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• In order to demonstrate that the nucleic acid-binding activities of vimentin are dictated by its Arg-rich N-terminal head domain, this was cut off at position Lys96 with lysine-specific endoproteinase and analysed for its capacity to associate with a variety of synthetic and naturally occurring nucleic acids. The isolated polypeptide (vim NT) showed a preference for single-stranded (ss) polynucleotides, particularly for ssDNAs of high G-content. A comparison of the sequence and predicted secondary structure of vim NT with that of two prokaryotic ssDNA-binding proteins, G5P and G32P of bacteriophages fd and T4, respectively, revealed that the nucleic acid-binding region of all three polypeptides is almost entirely in the beta-conformation and characterized by a very similar distribution of aromatic amino acid residues. A partial sequence of vim NT can be folded into the same beta-loop structure as the DNA-binding wing of G5P of bacteriophage fd and related viruses. As in the case of G5P, nitration of the Tyr residues with tetranitromethane was blocked by single-stranded nucleic acids. This and spectroscopic data indicate intercalation of the Tyr aromatic ring systems between the bases of the nucleic acids and thus the contribution of a stacking component to the binding reaction. The binding was accompanied by significant changes in the ultraviolet absorption spectra of both vim NT and single-stranded nucleic acids. Upon mixing of vim NT with nucleic acids, massive precipitation of the reactants occurred, followed by the quick rearrangement of the aggregates with the formation of specific and soluble association products. Even at very high ionic strengths, at which no electrostatic reaction should be expected, a distinct fraction of vim NT incorporated naturally occurring ssRNAs and ssDNAs into fast sedimenting complexes, suggesting co-operative interaction of the polypeptide with the nucleic acids. In electron microscopy, the complexes obtained from 28 S rRNA appeared as networks of extended nucleic acid strands densely covered with vim NT, in contrast to the compact random coils of uncomplexed RNA. The networks produced from fd DNA were heterogeneous in appearance and their nucleoprotein strands in rare cases were very similar to the rod-like structures of G5P-fd DNA complexes.