A DNA duplex containing the primary acrolein adduct, 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purin-10(3H)-one (2), of deoxyguanosine in a 5'-CpG sequence context spontaneously but reversibly formed an interchain cross-link with the exocyclic amino group of deoxyguanosine in the opposing chain. The linkage was sufficiently stable that the cross-linked duplex could be isolated by HPLC and characterized by MALDI-TOF mass spectrometry. Enzymatic degradation gave bis-nucleoside 6, which was independently prepared by direct reaction of 2 with dGuo.

Expression of fission yeast Pho1 acid phosphatase is repressed during growth in phosphate-rich medium. Repression is mediated by transcription of the prt locus upstream of pho1 to produce a long noncoding (lnc) prt RNA. Repression is also governed by RNA polymerase II CTD phosphorylation status, whereby inability to place a Ser7-PO 4 mark (as in S7A) derepresses Pho1 expression, and inability to place a Thr4-PO 4 mark (as in T4A) hyper-represses Pho1 in phosphate replete cells. Here we find that basal pho1 expression from the prt-pho1 locus is inversely correlated with the activity of the prt promoter, which resides in a 110-nucleotide DNA segment preceding the prt transcription start site. CTD mutations S7A and T4A had no effect on the activity of the prt promoter or the pho1 promoter, suggesting that S7A and T4A affect post-initiation events in prt lncRNA synthesis that make it less and more repressive of pho1, respectively. prt lncRNA contains clusters of DSR (determinant of selective removal) sequences recognized by the YTH-domain-containing protein Mmi1. Altering the nucleobase sequence of two DSR clusters in the prt lncRNA caused hyper-repression of pho1 in phosphate replete cells, concomitant with increased levels of the prt transcript. The isolated Mmi1 YTH domain binds to RNAs with single or tandem DSR elements, to the latter in a noncooperative fashion. We report the 1.75 Å crystal structure of the Mmi1 YTH domain and provide evidence that Mmi1 recognizes DSR RNA via a binding mode distinct from that of structurally homologous YTH proteins that recognize m 6 Amodified RNA.

The carboxy-terminal domain (CTD) code encrypted within the Y1S2P3T4S5P6S7heptad repeats of RNA polymerase II (Pol2) is deeply rooted in eukaryal biology. Key steps to deciphering the code are identifying the events in gene expression that are governed by individual “letters” and then defining a vocabulary of multiletter “words” and their meaning. Thr4 and Ser7 exert opposite effects on the fission yeastpho1gene, expression of which is repressed under phosphate-replete conditions by transcription of an upstream flanking long noncoding RNA (lncRNA). Here we attribute the derepression ofpho1by a CTD-S7Amutation to precocious termination of lncRNA synthesis, an effect that is erased by mutations of cleavage-polyadenylation factor (CPF) subunits Ctf1, Ssu72, Ppn1, Swd22, and Dis2 and termination factor Rhn1. By contrast, a CTD-T4Amutation hyperrepressespho1, as do CPF subunit and Rhn1 mutations, implying thatT4Areduces lncRNA termination. Moreover, CTD-T4Ais synthetically lethal withppn1∆ andswd22∆, signifying that Thr4 and the Ppn1•Swd22 module play important, functionally redundant roles in promoting Pol2 termination. We find that Ppn1 and Swd22 become essential for viability when the CTD array is curtailed and thatS7Aovercomes the need for Ppn1•Swd22 in the short CTD context. Mutational synergies highlight redundant essential functions of (i) Ppn1•Swd22 and Rhn1, (ii) Ppn1•Swd22 and Ctf1, and (iii) Ssu72 and Dis2 phosphatases. CTD allelesY1F,S2A, andT4Ahave overlapping synthetic lethalities withppn1∆ andswd22∆, suggesting that Tyr1-Ser2-Thr4 form a three-letter CTD word that abets termination, with Rhn1 being a likely “reader” of this word.

Acrolein is a bifunctional electrophile, present as an ubiquitous environmental pollutant and an endogenous cellular product of lipid peroxidation. Reaction of acrolein with deoxyguanosine produces two regioisomeric DNA adducts, specifically gamma-hydroxypropanodeoxyguanosine (gamma-HOPdG) and alpha-hydroxypropanodeoxyguanosine (alpha-HOPdG). While previous investigations have focused on the major gamma-HOPdG adduct, little is known about the properties of the minor alpha-HOPdG adduct. Therefore, this comparative investigation has assessed the following: the ability of each adduct to undergo secondary chemical reactions with biomolecules to form various cross-linked species, in vitro translesion DNA synthesis, and mutagenic properties, following replication in mammalian cells. In contrast to gamma-HOPdG, which is capable of forming DNA-DNA, DNA-peptide, and DNA-protein cross-links, alpha-HOPdG did not form any of these cross-linked species. These results can be attributed to the inability of the alpha-HOPdG adduct to undergo ring opening, whereas the gamma-HOPdG adduct forms the ring open, acyclic N(2) oxopropyl in duplex DNA, which readily reacts with nucleophilic functions. Consistent with this interpretation, when polymerase eta replication bypass of DNA containing alpha-HOPdG was assayed, this lesion posed a stronger block to replication than the gamma-HOPdG adduct, closely resembling the results for polymerase eta bypass of propanodeoxyguanosine in which the exocyclic adduct remains permanently ring-closed. Cellular replication and mutagenesis assays in COS-7 cells using single-stranded DNA containing a site specific alpha-HOPdG revealed that this adduct was significantly mutagenic, yielding a nearly identical frequency and spectrum of mutations as compared with the gamma-HOPdG adduct.

Initiation of inhalation anthrax is believed to involve phagocytosis of Bacillus anthracis spores by alveolar macrophages, followed by spore germination within the phagolysosome. In order to establish a systemic infection, it is predicted that bacilli then escape from the macrophage and replicate extracellularly. Mechanisms utilized by B. anthracis to escape from the macrophage are not well characterized, but a role for anthrax toxin has been proposed. Here we report the isolation of an anthrax toxin-resistant cell line (R3D) following chemical mutagenesis of toxin-sensitive RAW 264.7 murine macrophage cells. Both R3D and RAW 264.7 cells phagocytize spores of a B. anthracis Sterne strain. However, RAW 264.7 cells are killed following spore challenge, whereas R3D cells survive. Resistance to toxin and spore challenge correlates with loss of expression of anthrax toxin receptor 2 (ANTXR2/CMG-2). When R3D cells are complemented with cDNA encoding either murine ANTXR2 or human anthrax toxin receptor 1 (ANTXR1/TEM-8), toxin and spore challenge susceptibility are restored, indicating that over-expression of either ANTXR can confer susceptibility to anthrax spore challenge. Taken together, these results indicate that anthrax toxin expression by the germinated spore enables B. anthracis killing of the macrophage from within.

The primary structure and phosphorylation pattern of the tandem Y 1 S 2 P 3 T 4 S 5 P 6 S 7 repeats of the RNA polymerase II carboxyl-terminal domain (CTD) comprise an informational code that coordinates transcription, chromatin modification, and RNA processing. To gauge the contributions of individual CTD coding "letters" to gene expression, we analyzed the poly(A) + transcriptomes of fission yeast mutants that lack each of the four inessential CTD phosphoacceptors: Tyr1, Ser2, Thr4, and Ser7. There was a hierarchy of CTD mutational effects with respect to the number of dysregulated protein-coding RNAs, with S2A (n = 227) >> Y1F (n = 71) > S7A (n = 58) >> T4A (n = 7). The majority of the protein-coding RNAs affected in Y1F cells were coordinately affected by S2A, suggesting that Tyr1-Ser2 constitutes a two-letter code "word." Y1F and S2A elicited increased expression of genes encoding proteins involved in iron uptake (Frp1, Fip1, Fio1, Str3, Str1, Sib1), without affecting the expression of the genes that repress the iron regulon, implying that Tyr1-Ser2 transduces a repressive signal. Y1F and S2A cells had increased levels of ferric reductase activity and were hypersensitive to phleomycin, indicative of elevated intracellular iron. The T4A and S7A mutations had opposing effects on the phosphate response pathway. T4A reduced the expression of two genes encoding proteins involved in phosphate acquisition (the Pho1 acid phosphatase and the phosphate transporter SPBC8E4.01c), without affecting the expression of known genes that regulate the phosphate response pathway, whereas S7A increased pho1 + expression. These results highlight specific cellular gene expression programs that are responsive to distinct CTD cues.iron homeostasis | phosphate homeostasis | transcription profiling T he carboxyl-terminal domain (CTD) of the Rpb1 subunit of RNA polymerase II (Pol II) consists of tandemly repeated heptapeptides of consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 . The inherently plastic CTD structure is modulated by phosphorylation of the Tyr1, Ser2, Thr4, Ser5, and Ser7 residues and by cis-trans isomerization of the prolines (1, 2). With as many as 128 n potential CTD primary structures (where n is the number of heptads), the CTD provides information about the state of the transcription machinery-a CTD code-that is "read" by CTD receptor proteins that control transcription, modify chromatin structure, and catalyze or regulate mRNA capping, splicing, and polyadenylation (1, 2).Basic informational rules that govern the CTD code have been elucidated by genetically manipulating the composition and structure of the Rpb1 CTD in the fission yeast Schizosaccharomyces pombe (3-7). By introducing alanines and conservative mutations in lieu of Tyr1, Ser2, Pro3, Thr4, Ser5, Pro6, and Ser7 of every consensus heptad of a fully functional Rpb1 CTD array (comprising 14 consensus heptad repeats linked to the body of Rpb1 by a "rump" consisting of four degenerate heptads), we determined that: (i) Tyr1, Pro3, Ser5, and Pro6 are essential ...

The thermal cis-trans isomerization of methyl orange (i.e., sodium 4-[4'-(dimethylamino)phenylazo]benzenesulfonate) in acidic aqueous solutions has been investigated by means of laser-flash photolysis techniques. The thermal cis-trans isomerization is found to be catalyzed by general acids and general bases. Catalysis is attributed to acid/base-assisted tautomerization of cis-ammonium ions (formed by rapid protonation of cis-methyl orange generated upon photochemically induced trans-cis isomerization) into cis-azonium ions. The latter can easily isomerize via rotation around the -N=N- bond as a result of the concomitant decrease in the double bond character. Furthermore, the acidity of cis-ammonium ions is estimated to be significantly lower than that reported for the trans isomers (pK(a) values are 5.0 and 2.70-2.87, respectively). This result is attributed to a decrease in resonance interactions of the two aryl rings in the cis isomer compared with the trans form due to the nonplanar conformation of the former.

Fission yeast phosphate acquisition genes pho1, pho84, and tgp1 are repressed in phosphate-rich medium by transcription of upstream lncRNAs. Here, we show that phosphate homeostasis is subject to metabolite control by inositol pyrophosphates (IPPs), exerted through the 3′-processing/termination machinery and the Pol2 CTD code. Increasing IP8 (via Asp1 IPP pyrophosphatase mutation) de-represses the PHO regulon and leads to precocious termination of prt lncRNA synthesis. pho1 de-repression by IP8 depends on cleavage-polyadenylation factor (CPF) subunits, termination factor Rhn1, and the Thr4 letter of the CTD code. pho1 de-repression by mutation of the Ser7 CTD letter depends on IP8. Simultaneous inactivation of the Asp1 and Aps1 IPP pyrophosphatases is lethal, but this lethality is suppressed by mutations of CPF subunits Ppn1, Swd22, Ssu72, and Ctf1 and CTD mutation T4A. Failure to synthesize IP8 (via Asp1 IPP kinase mutation) results in pho1 hyper-repression. Synthetic lethality of asp1Δ with Ppn1, Swd22, and Ssu72 mutations argues that IP8 plays an important role in essential 3′-processing/termination events, albeit in a manner genetically redundant to CPF. Transcriptional profiling delineates an IPP-responsive regulon composed of genes overexpressed when IP8 levels are increased. Our results establish a novel role for IPPs in cell physiology.