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[Image: see text] 1,N(6)-ethenoadenine (εA) is a mutagenic lesion and biomarker observed in numerous cancerous tissues. Two pathways are responsible for its repair: base excision repair (BER) and direct reversal repair (DRR). Alkyladenine DNA glycosylase (AAG) is the primary enzyme that excises εA in BER, generating stable intermediates that are processed by downstream enzymes. For DRR, the Fe(II)/α-ketoglutarate-dependent ALKBH2 enzyme repairs εA by direct conversion of εA to A. While the molecular mechanism of each enzyme is well understood on unpackaged duplex DNA, less is known about their actions on packaged DNA. The nucleosome core particle (NCP) forms the minimal packaging unit of DNA in eukaryotic organisms and is composed of 145–147 base pairs wrapped around a core of eight histone proteins. In this work, we investigated the activity of AAG and ALKBH2 on εA lesions globally distributed at positions throughout a strongly positioned NCP. Overall, we examined the repair of εA at 23 unique locations in packaged DNA. We observed a strong correlation between rotational positioning of εA and AAG activity but not ALKBH2 activity. ALKBH2 was more effective than AAG at repairing occluded εA lesions, but only AAG was capable of full repair of any εA in the NCP. However, notable exceptions to these trends were observed, highlighting the complexity of the NCP as a substrate for DNA repair. Modeling of binding of the repair enzymes to NCPs revealed that some of these observations can be explained by steric interference caused by DNA packaging. Specifically, interactions between ALKBH2 and the histone proteins obstruct binding to DNA, which leads to diminished activity. Taken together, these results support in vivo observations of alkylation damage profiles and contribute to our understanding of mutational hotspots.

It has been predicted that 30-80% of archaeal genomes remain annotated as hypothetical proteins with no assigned gene function. Further, many archaeal organisms are difficult to grow or are unculturable. To overcome these technical and experimental hurdles, we have developed a high-throughput functional genomics screen that utilizes capillary electrophoresis (CE) to identify nucleic acid modifying enzymes based on activity rather than sequence homology. Here, we describe a functional genomics screening workflow to find DNA modifying enzyme activities encoded by the hyperthermophile Thermococcus kodakarensis (T. kodakarensis). Large DNA insert fosmid libraries representing a ∼5-fold average coverage of the T. kodakarensis genome were prepared in E. coli. RNA-seq showed a high fraction (84%) of T. kodakarensis genes were transcribed in E. coli despite differences in promoter structure and translational machinery. Our high-throughput screening workflow used fluorescently labeled DNA substrates directly in heat-treated lysates of fosmid clones with capillary electrophoresis detection of reaction products. Using this method, we identified both a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and a novel AP lyase DNA repair enzyme family (termed 'TK0353') found only in a small subset of Thermococcales. The screening methodology described provides a fast and efficient way to explore the T. kodakarensis genome for a variety of nucleic acid modifying activities and may have implications for similar exploration of enzymes and pathways that underlie core cellular processes in other Archaea. IMPORTANCE This study provides a rapid, simple, high-throughput method to discover novel archaeal nucleic acid modifying enzymes by utilizing a fosmid genomic library, next-generation sequencing and capillary electrophoresis. The method described here provides details necessary to create 384-well fosmid library plates from Thermococcus kodakarensis genomic DNA, sequence 384-well fosmids plates using Illumina next generation sequencing and perform high-throughput functional read-out assays using capillary electrophoresis to identify a variety of nucleic acid modifying activities including DNA cleavage and ligation. We used this approach to identify a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and identify a novel AP lyase enzyme (TK0353) that lacks sequence homology to known nucleic acid modifying enzymes.

A recently discovered post-translational modification of histone proteins is the irreversible proteolytic clipping of the histone N-terminal tail domains. This modification is involved in the regulation of various biological processes, including the DNA damage response. In this work, we used chemical footprinting to characterize the structural alterations to nucleosome core particles (NCPs) that result from a lack of a histone H2B or H3 tail. We also examine the influence of these histone tails on excision of the mutagenic lesion 1,N6-ethenoadenine (eA) by the repair enzyme alkyladenine DNA glycosylase. We found that the absence of the H2B or H3 tail results in altered DNA periodicity relative to that of native NCPs. We correlated these structural alterations to eA excision by utilizing a global analysis of 21 eA sites in NCPs and unincorporated duplex DNA. In comparison to native NCPs, there is enhanced excision of eA in tailless H2B NCPs in regions that undergo DNA unwrapping. This enhanced excision is not observed for tailless H3 NCPs; rather, excision is inhibited in more static areas of the NCP not prone to unwrapping. Our results support in vivo observations of alkylation damage profiles and the potential role of tail clipping as a mechanism for overcoming physical obstructions caused by packaging in NCPs but also reveal the potential inhibition of repair by tail clipping in some locations. Taken together, these results further our understanding of how base excision repair can be facilitated or diminished by histone tail removal and contribute to our understanding of the underlying mechanism that leads to mutational hot spots.

The base excision repair (BER) pathway removes modified nucleobases that can be deleterious to an organism. BER is initiated by a glycosylase, which finds and removes these modified nucleobases. Most of the characterization of glycosylase activity has been conducted in the context of DNA oligomer substrates. However, DNA within eukaryotic organisms exists in a packaged environment with the basic unit of organization being the nucleosome core particle (NCP). The NCP is a complex substrate for repair in which a variety of factors can influence glycosylase activity. In this Review, we focus on the geometric positioning of modified nucleobases in an NCP and the consequences on glycosylase activity and initiating BER.

DNA is comprised of chemically reactive nucleobases that exist under a constant barrage from damaging agents. Failure to repair chemical modifications to these nucleobases can result in mutations that can cause various diseases, including cancer. Fortunately, the base excision repair (BER) pathway can repair modified nucleobases and prevent these deleterious mutations. However, this pathway can be hindered through several mechanisms. For instance, mutations to the enzymes in the BER pathway have been identified in cancers. Biochemical characterisation of these mutants has elucidated various mechanisms that inhibit their activity. Furthermore, the packaging of DNA into chromatin poses another obstacle to the ability of BER enzymes to function properly. Investigations of BER in the base unit of chromatin, the nucleosome core particle (NCP), have revealed that the NCP acts as a complex substrate for BER enzymes. The constituent proteins of the NCP, the histones, also have variants that can further impact the structure of the NCP and may modulate access of enzymes to the packaged DNA. These histone variants have also displayed significant clinical effects both in carcinogenesis and patient prognosis. This review focuses on the underlying molecular mechanisms that present obstacles to BER and the relationship of these obstacles to cancer. In addition, several chemotherapeutics induce DNA damage that can be repaired by the BER pathway and understanding obstacles to BER can inform how resistance and/or sensitivity to these therapies may occur. With the understanding of these molecular mechanisms, current chemotherapeutic treatment regiments may be improved, and future therapies developed.

PTEN, a tumor suppressor among the most commonly mutated proteins in human cancer, is recognized to be both a protein phosphatase and a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) 3-phosphatase. Previous work [Maehama and Dixon, J. Biol. Chem. 273 (1998) 13375–13378] has led to a consensus that inositol phosphates are not physiologically relevant substrates for PTEN. In contrast, we demonstrate that PTEN is an active inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P 5 ) 3-phosphatase when expressed and purified from bacteria or HEK cells. Kinetic data indicate Ins(1,3,4,5,6)P 5 ( K m =7.1 μM) and PtdIns(3,4,5)P 3 ( K m =26 μM) compete for PTEN in vivo. Transient transfection of HEK cells with PTEN decreased Ins(1,3,4,5,6)P 5 levels. We discuss the physiological significance of these studies in relation to recent work showing that dephosphorylation of Ins(1,3,4,5,6)P 5 to inositol 1,4,5,6-tetrakisphosphate is a cell signaling event.

The objective of this study was to determine the effects on animal performance and rumen digestion characteristics of supplementing cattle grazing autumn grass offered at two levels of allowance, with concentrates formulated from different carbohydrate sources. Seventy-two steers (494 (s.e. 38·4) kg) were assigned to one of six treatments which were arranged in a two grass allowances (5·5 or 11 kg grass dry matter (DM) per head per day) by three concentrate types (starch, starch + fibre or fibre-based diets) factorial design. The experiment commenced on 22 August and had a mean duration of 84 days. Concentrates were formulated to have similar metabolizable energy and crude protein and animals were offered 5 kg per head daily. Grass allowances were offered daily and concentrates were offered individually. Six rumen fistulated steers were used concurrently in a Latin square design to determine the effect of the diets on rumen digestion characteristics. Grass and concentrates degradabilities were measured using the nylon bag technique. The concentrate DM was degraded at 0·189, 0·102 and 0·094 per h for the starch, starch + fibre and fibre-based diets, respectively. Concentrate carbohydrate source and grass allowance did not affect (P> 0·05) degradation rate of grass DM or nitrogen in the rumen, rumen fluid pH, rumen fluid concentration of volatile fatty acids, total lactic acid or ammonia concentration or plasma concentration of glucose, urea or beta-hydroxybutyrate. Animals offered the low grass allowance had larger (P< 0·05) diurnal variation in the rumen fluid acetate: propionate ratio than those offered the high grass allowance. Increasing grass allowance increased (P< 0·01) carcass gain whereas concentrate type had no effect. There was no interaction between concentrate carbohydrate source and grass allowance on any of the production variables measured. It is concluded that the rumen environment in cattle grazing autumn grass buffered the effects of concentrate carbohydrate degradability such that similar productivity was achieved with each concentrate.

Saiardi et al. (Saiardi, A., Erdjument-Bromage, H., Snowman, A., Tempst, P., and Snyder, S. H. (1999) Curr. Biol. 9, 1323-1326) previously described the cloning of a kinase from yeast and two kinases from mammals (types 1 and 2), which phosphorylate inositol hexakisphosphate (InsP(6)) to diphosphoinositol pentakisphosphate, a "high energy" candidate regulator of cellular trafficking. We have now studied the significance of InsP(6) kinase activity in Saccharomyces cerevisiae by disrupting the kinase gene. These ip6kDelta cells grew more slowly, their levels of diphosphoinositol polyphosphates were 60-80% lower than wild-type cells, and the cells contained abnormally small and fragmented vacuoles. Novel activities of the mammalian and yeast InsP(6) kinases were identified; inositol pentakisphosphate (InsP(5)) was phosphorylated to diphosphoinositol tetrakisphosphate (PP-InsP(4)), which was further metabolized to a novel compound, tentatively identified as bis-diphosphoinositol trisphosphate. The latter is a new substrate for human diphosphoinositol polyphosphate phosphohydrolase. Kinetic parameters for the mammalian type 1 kinase indicate that InsP(5) (K(m) = 1.2 micrometer) and InsP(6) (K(m) = 6.7 micrometer) compete for phosphorylation in vivo. This is the first time a PP-InsP(4) synthase has been identified. The mammalian type 2 kinase and the yeast kinase are more specialized for the phosphorylation of InsP(6). Synthesis of the diphosphorylated inositol phosphates is thus revealed to be more complex and interdependent than previously envisaged.

The turnover of the “high energy” diphosphoinositol polyphosphates by Ca2+- and cyclic nucleotide-modulated enzymes is considered a regulatory, molecular switching activity. Target processes may include intracellular trafficking. Following our earlier identification of a prototype humandiphosphoinositol-polyphosphatephosphohydrolase (hDIPP1), we now describe new 21-kDa human isoforms, hDIPP2α and hDIPP2β, distinguished from each other solely by hDIPP2β possessing one additional amino acid (Gln86). Candidate DIPP2α and DIPP2β homologues in rat and mouse were also identified. The rank order for catalytic activity is hDIPP1 > hDIPP2α > hDIPP2β. Differential expression of hDIPP isoforms may provide flexibility in response times of the molecular switches. The 76% identity between hDIPP1 and the hDIPP2s includes conservation of an emerging signature sequence, namely, a Nudt (MutT) motif with a GX 2GX 6G carboxy extension. Northern and Western analyses indicate expression of hDIPP2s is broad but atypically controlled; these proteins are translated from multiple mRNAs that differ in the length of the 3′-untranslated region because of utilization of an array of alternative (canonical and noncanonical) polyadenylation signals. Thus, cells can recruit sophisticated molecular processes to regulate diphosphoinositol polyphosphate turnover.

The ARGRIII gene of Saccharomyces cerevisiae encodes a transcriptional regulator that also has inositol polyphosphate multikinase (ipmk) activity [Saiardi et al. (1999) Curr. Biol. 9, 1323-1326]. To investigate how inositol phosphates regulate gene expression, we disrupted the ARGRIII gene. This mutation impaired nuclear mRNA export, slowed cell growth, increased cellular [InsP(3)] 170-fold and decreased [InsP(6)] 100-fold, indicating reduced phosphorylation of InsP(3) to InsP(6). Levels of diphosphoinositol polyphosphates were decreased much less dramatically than was InsP(6). Low levels of InsP(6), and considerable quantities of Ins(1,3,4,5)P(4), were synthesized by an ipmk-independent route. Transcriptional control by ipmk reflects that it is a pivotal regulator of nuclear mRNA export via inositol phosphate metabolism.

We have derived the full-length sequences of the human and rat forms of the multiple inositol polyphosphate phosphatase (MIPP); their structural and functional comparison with a chick histidine acid phosphatase (HiPER1) has revealed new information: (1) MIPP is approximately 50% identical to HiPER1, but the ER-targeting domains are divergent; (2) MIPP appears to share the catalytic requirement of histidine acid phosphatases, namely, a C-terminal His residue remote from the RHGxRxP catalytic motif; (3) rat MIPP mRNA is up-regulated during chondrocyte hypertrophy. The latter observation provides a context for proposing that MIPP may aid bone mineralization and salvage the inositol moiety prior to apoptosis.

Summary The distribution of acetyl-CoA carboxylase between the epidermal and mesophyll layers of leek leaves was determined. The specific activity of acetyl-CoA carboxylase was approximately three-fold higher in extracts of the epidermis than in those from the mesophyll layer. Four prominent biotinylated polypeptides of approximate molecular masses 240, 85, 37, and 35 ku (kDa) were detected in leaf extracts. The biotincontaining subunit of the homomeric acetyl-CoA carboxylase, the 240-ku biotinylated polypeptide, accumulates exclusively in the epidermis of leek leaves. Three of the subunits of the heteromeric acetyl-CoA carboxylase (the biotin carboxyl carrier, the biotin carboxylase, and s-subunit of the carboxyltransferase) were equally abundant in leek mesophyll and epidermal tissues. These data indicate that leek leaves contain two structurally distinct isozymes of acetyl-CoA carboxylase, one of which, the homomeric form, is restricted to the epidermis.

The effects on rumen fermentation and blood metabolites when barley or cane molasses were offered as isoenergetic and isonitrogenous supplements at 610 g kg−1 of total dietary dry matter (DM) intake were studied in steers fed grass silage. Six rumen fistulated steers were used in a 2 (supplements) × 2 (periods, 28 days duration) cross-over experiment. The animals were offered 35.2 g supplement DM and 22 g silage DM kg−1 bodyweight0.75 daily at 08:30 and 16:30. Rumen fluid samples were collected from all animals on Days 27 and 28 of each period before the morning feed and 1, 2, 3, 6, 8, 9, 10, 14 and 24 h thereafter. Blood samples were collected from each steer on Day 26, before the morning feed and 1 and 3 h thereafter. Mean rumen pH (6.9 vs. 6.9), concentration of total volatile fatty acids (71.2 vs. 71.7 mmol−1) and molar proportion of propionate (15.8 vs. 16.6) were similar for both supplements. The molasses-based supplement resulted in a lower (P < 0.01) rumen concentration (mg dl−1) of ammonia-N (5.6 vs. 9.3) and l-lactate (26.2 vs. 49.3), and as proportions of total volatile fatty acids, lower (P < 0.01) acetate (58.4 vs. 66.5) and higher (P < 0.01) butyrate (23.0 vs. 14.0) than the barley-based supplement. Type of supplement did not affect the concentration of haemoglobin in blood. Plasma glucose concentration was higher (P < 0.01) before the morning feed and mean plasma urea-N was lower (P < 0.06) in animals offered the molasses-based supplement. Sampling time significantly affected all blood metabolites and rumen fermentation variables measured. It is concluded that under the conditions of this study, plasma glucose and urea-N concentrations and the pattern of rumen fermentation differed between barley and molasses-based supplements to grass silage.

Diphosphoinositol pentakisphosphate (PP-InsP5 or 'InsP7') and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4 or 'InsP8') are the most highly phosphorylated members of the inositol-based cell signaling family. We have purified a rat hepatic diphosphoinositol polyphosphate phosphohydrolase (DIPP) that cleaves a beta-phosphate from the diphosphate groups in PP-InsP5 (Km = 340 nM) and [PP]2-InsP4 (Km = 34 nM). Inositol hexakisphophate (InsP6) was not a substrate, but it inhibited metabolism of both [PP]2-InsP4 and PP-InsP5 (IC50 = 0.2 and 3 microM, respectively). Microsequencing of DIPP revealed a 'MutT' domain, which in other contexts guards cellular integrity by dephosphorylating 8-oxo-dGTP, which causes AT to CG transversion mutations. The MutT domain also metabolizes some nucleoside phosphates that may play roles in signal transduction. The rat DIPP MutT domain is conserved in a novel recombinant human uterine DIPP. The nucleotide sequence of the human DIPP cDNA was aligned to chromosome 6; the candidate gene contains at least four exons. The dependence of DIPP's catalytic activity upon its MutT domain was confirmed by mutagenesis of a conserved glutamate residue. DIPP's low molecular size, Mg2+ dependency and catalytic preference for phosphoanhydride bonds are also features of other MutT-type proteins. Because overlapping substrate specificity is a feature of this class of proteins, our data provide new directions for future studies of higher inositol phosphates.

At least six biotinylated polypeptides can be detected in extracts from plants (1). These polypeptides are approximately 240 kDa, 120 kDa, 75 kDa, 60 kDa, 39 kDa and 34 kDa in size. Furthermore, the biotin enzymes, acetyl-CoA carboxylase (ACCase), propionyl-CoA carboxylase (PCCase), methylcrotonyl-CoA carboxylase (MCCase), pyruvate carboxylase (1) and geranoyl-CoA carboxylase (GCCase) are detectable in extracts from plants, We have undertaken biochemical and molecular biological approaches to identify and characterize the enzymes these polypeptides represent.

Acetyl-CoA carboxylase (ACCase) catalyzes the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA. Classically this reaction is considered to be the first, rate-limiting reaction of de novo fatty acid biosynthesis. In contrast to all other organisms, which synthesize fatty acids in the cytosol, plants are unique in that de novo fatty acid biosynthesis occurs almost exclusively in plastids. In addition, plants utilize malonyl-CoA for the synthesis of a number of secondary metabolites, including flavonoids, very long chain fatty acids, stilbenoids, and many malonyl derivatives; these reactions are thought to occur in the cytosol of plant cells (1, 2, introduction of reference #3). To characterize how malonyl-CoA is generated from acetyl-CoA, in at least two compartments, we have been characterizing ACCase via a comprehensive study of biotinylated proteins. Our studies, and those from other labs, indicate that plants contain at least two structurally distinct ACCases. One form of ACCase has a biotin-containing subunit of approximately 240 kDa, and a second form has a biotin-containing polypeptide of about 60 kDa.

Enterotoxigenic Escherichia coli (ETEC) strains were readily identified in pure and mixed cultures with nonradioactive, digoxigenin-labeled DNA probes coding for heat-labile (LTI) and heat-stable (STaI, STaII, and STb) enterotoxins. Digoxigenin-labeled ETEC fragments were more sensitive than and exhibited less nonspecific background contamination than biotinylated ETEC probes.

The effects of grazed grass, grass silage, or concentrates on fatty acid composition and conjugated linoleic acid (cis-9, trans-11-18:2; CLA) concentrations of i.m. fat of steers fed to achieve similar carcass growth rates were investigated. Fifty steers were divided into 10 blocks based on body weight and assigned at random from within blocks to one of five dietary treatments. The experimental rations offered daily for 85 d preceding slaughter were 1) grass silage for ad libitum intake plus 4 kg of concentrate, 2) 8 kg of concentrate plus 1 kg of hay, 3) 6 kg of grazed grass DM plus 5 kg of concentrate, 4) 12 kg of grazed grass DM plus 2.5 kg concentrate, or 5) 22 kg of grazed grass DM. The concentration of polyunsaturated fatty acids (PUFA) in i.m. fat was higher (P < .05) for steers offered ration 5 than for those given any other ration. Decreasing the proportion of concentrate in the diet, which effectively increased grass intake, caused a linear decrease in the concentration of i.m. saturated fatty acids (SFA) (P < .01) and in the n-6:n-3 PUFA ratio (P < .001) and a linear increase in the PUFA:SFA ratio (P < .01) and the conjugated linoleic acid concentration (P < .001). The data indicate that i.m. fatty acid composition of beef can be improved from a human health perspective by inclusion of grass in the diet.

SUMMARY1. Daily allowances of 1·37 kg of concentrates, containing either 8·9 (treatment A), 13·6 (B), 19·1 (C) or 29·2% crude protein (D) were given to 40 individually-fed steers eating long barley straw to appetite over a 112-day winter feeding period. Voluntary straw intake on treatment B was 25% higher than that on treatment A. There were no signiicant differences in straw intake between treat-ments B, C, and D. Mean daily live-weight gains (kg) for the treatment groups were A, −0·22; B, 0·05; C, 0·04; D, 0·15. When the animals were turned out to pastore, group A showed very rapid compensatory growth, and it was concluded that this was mainly due to differences between the groups in their changes in alimentary tract contents. Vitamin A and D supplementation did not affect straw intake or performance.2. When the above supplements were given to steers in metabolism crates receiving barley straw in restricted or unrestricted quantities, it was found that an increase in the crude-protein content of the supplement significantly increased the digestibility of the organic matter, crude fibre and crude protein of the whole diet when straw intake was restricted, and significantly increased the digestibility of crude fibre and crude protein when straw was unrestricted.3. It was concluded that voluntary straw intake was limited, at the lowest level of supplementary protein, by a deficiency of absorbed protein. At the higher levels of supplementary protein, intake was probably limited by the capacity of the alimentary tract.

The Arg82 gene of Saccharomyces cerevisiae encodes a transcriptional regulator that phosphorylates inositol 1,4,5-trisphosphate [Saiardi et al. (1999) Curr. Biol. 9, 1323–1326]. However, some controversy has surrounded the nature of the reaction products. We now show that Arg82 phosphorylates inositol 1,3,4,5-tetrakisphosphate to inositol pentakisphosphate, which is itself converted to two isomers of diphosphoinositol tetrakisphosphate, one of which has never previously been identified. One of the diphosphoinositol phosphates was further phosphorylated by a yeast cell lysate. We propose that Arg82 is an ancestral precursor of two distinct and specific enzyme families: inositol 1,4,5-trisphosphate kinases and diphosphoinositol polyphosphate synthases.