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The transcription factor NF-B, a central regulator of immunity, is subject to regulation by redox changes. We now report that cysteine-179 of the inhibitory B kinase (IKK) ␤-subunit of the IKK signalosome is a central target for oxidative inactivation by means of S-glutathionylation. S-glutathionylation of IKK-␤ Cys-179 is reversed by glutaredoxin (GRX), which restores kinase activity.

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Mitochondria infiltrate leading edge lamellipodia, increasing local mitochondrial mass and relative ATP concentration. AMPK regulates infiltration of mitochondria into the leading edge of 2D lamellipodia and 3D invadopodia, coupling local metabolic sensing to subcellular targeting of mitochondria during cell movement.

In order to study control ofDNA synthesis in complex eukaryotic systems, one would like to examine a single replicon whose time ofsynthesis within the S period is known. Certain cell lines contain amplified sequences that appear to replicate synchronously with respect to one another and therefore behave as ifthey were composed of one or a few replicon types (1, 2). These sequences are found in a variety of methotrexate (MTX)-resistant mouse and hamster cell lines, and arise through the amplification of a large segment which includes the gene for dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase;

All known DNA and RNA polymerases catalyze the formation of phosphodiester bonds in a 5′ to 3′ direction, suggesting this property is a fundamental feature of maintaining and dispersing genetic information. The tRNA His guanylyltransferase (Thg1) is a member of a unique enzyme family whose members catalyze an unprecedented reaction in biology: 3′-5′ addition of nucleotides to nucleic acid substrates. The 2.3-Å crystal structure of human THG1 (hTHG1) reported here shows that, despite the lack of sequence similarity, hTHG1 shares unexpected structural homology with canonical 5′-3′ DNA polymerases and adenylyl/guanylyl cyclases, two enzyme families known to use a two-metal-ion mechanism for catalysis. The ability of the same structural architecture to catalyze both 5′-3′ and 3′-5′ reactions raises important questions concerning selection of the 5′-3′ mechanism during the evolution of nucleotide polymerases. G-1 addition | reverse polymerase | tRNA modificationA ll nucleotide polymerases, including DNA and RNA polymerases, reverse transcriptase, and telomerase, catalyze nucleotide addition in the 5′ to 3′ direction. The reaction involves the nucleophilic attack of a polynucleotide terminal 3′-OH onto the α-phosphate of an incoming nucleotide, followed by release of the pyrophosphate moiety. Although the 5′ to 3′ direction has been adopted by all polymerases and transferases described to date, there is one notable exception: the enzyme tRNA His guanylyltransferase (Thg1). Thg1 catalyzes the highly unusual 3′-5′ addition of a single guanine to the 5′-end of tRNA His (1, 2). This reaction is an obligatory step in the maturation of this tRNA because the extra 5′ base, G −1 , constitutes a primary identity element for the aminoacyl-tRNA synthetase (HisRS) that attaches the amino acid histidine to the 3′-end of the tRNA (3-9). Thg1 is thus essential for maintaining the fidelity of protein synthesis. Consistent with the critical nature of the G −1 residue, THG1 is an essential gene in yeast and RNAi-mediated silencing of the Thg1 homolog in human cells results in severe cell-cycle progression and growth defects (2, 10, 11). Thg1 is widely conserved throughout eukarya, and Thg1 homologs are present in many archaea and bacteria.In eukarya, G −1 addition occurs opposite a universally conserved A 73 and thus is the result of a nontemplated 3′-5′ addition reaction. In addition, yeast Thg1 catalyzes a second reaction in vitro, extending tRNA substrates in the 3′-5′ direction in a template-directed manner driven by Watson-Crick pairing (12). Thg1 enzymes in archaea also catalyze template-dependent 3′-5′ addition, but do not catalyze nontemplated G −1 addition (13), suggesting that the templated 3′-5′ addition reaction likely represents an ancestral activity of the earliest Thg1 family members.The 3′-5′ addition of G −1 to tRNA His occurs via three chemical reactions, all catalyzed by Thg1 (2, 14) (Fig. 1). First, the 5′-monophosphorylated tRNA that results from RNase P cleavage of pre-tRNA His is activated using ATP, creating a 5...

We have developed a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) containing a 500-fold amplification of a 135-kilobase chromosomal DNA sequence. This sequence includes the gene for dihydrofolate reductase (tetrahydrofolate dehydrogenase, 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). The high copy number ofthe amplified sequence permits it to be visualized as a distinct series of restriction fragments in genomic digests separated on ethidium bromidestained agarose gels. Initiation of DNA replication in the amplified sequence was studied by radiolabeling DNA synthesized during the onset of S phase in synchronized CHOC 400 cells. Autoradiography of Southern blots of labeled genomic digests shows that DNA synthesis initiates in a small subset of the EcoRd fragments derived from the amplified units. These early labeled fragments are not synthesized at later times during S phase, when different subsets of fragments are synthesized. Regardless of the drug used to collect cells at the beginning of S phase, the replication pattern observed remains the same. These data suggest that replication of the amplified sequence initiates at specific sites within each repeated unit and proceeds in nonrandom order throughout the remainder of the sequence-i.e., that initiation of DNA synthesis in the chromosomes of mammalian cells is sequence specific.In a number of bacteriophages (1-3), animal cell viruses (4, 5), and bacteria (6, 7), initiation of DNA replication occurs from a genetically fixed nucleotide sequence termed the origin of replication. Molecular cloning has facilitated the isolation and analysis ofa number ofprokaryotic and viral origins and has allowed characterization of these regions with regard to their primary sequences (1-3, 5, 8, 9), possible secondary structures (2, 3), and probable interaction with specific DNA replication proteins (10, 11).In animal cells, considerable information supports a replicon model for chromosomal DNA replication (for review, see refs. 12 and 13) but, due to the complexity of the mammalian genome, initiation of DNA synthesis from fixed origins of DNA replication has not been demonstrated as yet. Recent approaches to the identification ofeukaryotic origins ofreplication have focused on the rescue by autonomous replication ofcertain recombinant plasmids containing eukaryotic genomic fragments after transfection into eukaryotic cells (for review, see ref. 14). However, the replication of extrachromosomal elements in these systems may not accurately reflect processes controlling chromosomal replication (15 (20). The nature of the remaining 100 kb in the unit repeated sequence is not known at present. Because of its high copy number in each cell, the entire amplified sequence can be visualized on ethidium bromide-stained gels as a series of 25-X30 distinct restriction fragments. The amplified dihydrofolate reductase genes present in CHOC 400 cells are located in several homogeneously staining chromosomal regions similar to those described for oth...