The Ku86 and XRCC4 proteins perform critical but poorly understood functions in the repair of DNA double-strand breaks. Both Ku 86- and XRCC4-deficient cells exhibit profound radiosensitivity and severe defects in V(D)J recombination, including excessive deletions at recombinant junctions. Previous workers have suggested that these phenomena may reflect defects in joining of the broken DNA ends or in protection of the ends from nucleases. However, end joining in XRCC4-deficient cells has not been examined. Here we show that joining of both matched and mismatched DNA ends occurs efficiently in XRCC4-deficient cells. Furthermore, analysis of junctions shows that XRCC4 is not required to protect the ends from degradation. However, nucleotide sequence analysis of junctions derived from joining of mismatched DNA ends in XRCC4-deficient cells revealed a strong preference for a junction containing a 7 nt homology. Similar results were obtained in Ku86-deficient cells. These data suggest that in the absence of XRCC4 or Ku86, joining is assisted by base pairing interactions, supporting the hypothesis that these proteins may participate in aligning or stabilizing intermediates in end joining.

PpsR from the anoxygenic phototrophic bacterium Rhodobacter sphaeroides has been known as an oxygenand light-dependent repressor of bacteriochlorophyll and carotenoid biosynthesis genes and puc operons involved in photosystem development. However, the putative PpsR-binding sites, TGTN 12 ACA, are also located upstream of numerous nonphotosystem genes, thus raising the possibility that the role of PpsR is broader. To characterize the PpsR regulon, transcriptome profiling was performed on the wild-type strain grown at high and low oxygen tensions, on the strain overproducing PpsR, and on the ppsR mutant. Transcriptome analysis showed that PpsR primarily regulates photosystem genes; the consensus PpsR binding sequence is TGTcN 10 gACA (lowercase letters indicate lesser conservation); the presence of two binding sites is required for repression in vivo. These findings explain why numerous single TGTN 12 ACA sequences are nonfunctional. In addition to photosystem genes, the hemC and hemE genes involved in the early steps of tetrapyrrole biosynthesis were identified as new direct targets of PpsR repression. Unexpectedly, PpsR was found to indirectly repress the puf and puhA operons encoding photosystem core proteins. The upstream regions of these operons contain no PpsR binding sites. Involvement in regulation of these operons suggests that PpsR functions as a master regulator of photosystem development. Upregulation of the puf and puhA operons that resulted from ppsR inactivation was sufficient to restore the ability to grow phototrophically to the prrA mutant. PrrA, the global redox-dependent activator, was previously considered indispensable for phototrophic growth. It is revealed that the PrrBA and AppA-PpsR systems, believed to work independently, in fact interact and coordinately regulate photosystem development.Rhodobacter sphaeroides is a facultatively phototrophic anoxygenic alphaproteobacterium. Under high oxygen tension, whether in the light or in the dark, this bacterium uses aerobic respiration for energy generation. When oxygen tension decreases, R. sphaeroides induces synthesis of photosynthetic apparatus, an alternate energy generation system functional under anoxic-light conditions (28,30,32,36). The photosynthetic apparatus is comprised of one type II photosystem (PS). Development of PS involves synthesis of photosynthetic pigments, bacteriochlorophyll (Bchl) and carotenoids (Crt), membrane proteins of the reaction center (RC), and two light-harvesting (LH) complexes as well as assembly factors. The RC and LH complexes are housed in the specialized intracytoplasmic membrane system. A decrease in oxygen tension triggers a significant increase in transcription of PS genes, which is required for PS development. Major regulatory components involved in oxygen control of PS development have been identified previously (1,44). However, precise roles of individual regulators and interactions between the regulatory pathways are not yet fully understood. This work is aimed at characterizing the role of one majo...

Acetaldehyde, the major ethanol metabolite that is far more toxic and reactive than ethanol, has been postulated to be responsible for alcohol-induced tissue and cell injury. This study was to examine whether facilitated acetaldehyde metabolism affects acetaldehyde-induced oxidative stress and apoptosis. Transgene-encoding human aldehyde dehydrogenase-2 (ALDH2), which converts acetaldehyde into acetate, was constructed under chicken ␤-actin promoter and transfected into human umbilical vein endothelial cells (HUVECs). Efficacy of ALDH2 transfection was verified using green fluorescent protein and ALDH2 enzymatic assay. Generation of reactive oxygen species (ROS) was measured using chloromethyl-2,7-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by 4,6-diamidino-2-phenylindoladihydrochloride fluorescence microscopy, quantitative DNA fragmentation, and caspase-3 assay. Acetaldehyde (0 -200 M) elicited ROS generation and apoptosis in HUVECs in a time-and concentration-dependent manner, associated with activation of the stress signal molecules ERK1/2 and p38 mitogen-activated protein (MAP) kinase. A close liner correlation was observed between the acetaldehyde-induced ROS generation and apoptosis. Interestingly, the acetaldehydeinduced ROS generation, apoptosis, activation of ERK1/2, and p38 MAP kinase were prevented by the ALDH2 transgene or antioxidant ␣-tocopherol. The involvement of ERK1/2 and p38 MAP kinase in acetaldehyde-induced apoptosis was confirmed by selective kinase inhibitors U0126, SB203580, and SB202190. Collectively, our data revealed that facilitation of acetaldehyde metabolism by ALDH2 transgene overexpression may prevent acetaldehyde-induced cell injury and activation of stress signals. These results indicated therapeutic potential of ALDH2 enzyme in the prevention and detoxification of acetaldehyde or alcohol-induced cell injury.Chronic alcohol consumption leads to cardiovascular complications such as endothelial dysfunction and alcoholic cardiomyopathy (1). Although several hypotheses have been speculated for alcohol-induced injury including direct/indirect toxicity of alcohol and accumulated fatty acid ethyl esters (2, 3), neither scenario has been fully validated by compelling clinical and experimental evidence. Acetaldehyde is the very first oxidized metabolic product of ethanol and is considered a candidate toxin for alcohol-induced tissue and cell injury. It is far more reactive than ethanol and may inhibit protein synthesis (4, 5). Our laboratory (6 -8) has shown that acetaldehyde interrupts cardiac excitation-contraction coupling and sarco(endo)plasmic reticulum Ca 2ϩ release function. Acetaldehyde has also been shown to form protein adducts leading to atherosclerotic vascular injury (9). Transgenic mice with cardiac overexpression of alcohol dehydrogenase (ADH) 1 displayed higher cardiac acetaldehyde levels associated with compromised heart function at whole heart and ventricular myocyte levels following alcohol intake (10 -12), suggesting that acetaldehyde may be one of t...

Cyclic dimeric GMP (c-di-GMP) is an important biofilm regulator that allosterically activates enzymes of exopolysaccharide biosynthesis. Proteobacterial genomes usually encode multiple GGDEF domain-containing diguanylate cyclases responsible for c-di-GMP synthesis. In contrast, only one conserved GGDEF domain protein, GdpS (for GGDEF domain protein from Staphylococcus), and a second protein with a highly modified GGDEF domain, GdpP, are present in the sequenced staphylococcal genomes. Here, we investigated the role of GdpS in biofilm formation in Staphylococcus epidermidis. Inactivation of gdpS impaired biofilm formation in medium supplemented with NaCl under static and flow-cell conditions, whereas gdpS overexpression complemented the mutation and enhanced wild-type biofilm development. GdpS increased production of the icaADBC-encoded exopolysaccharide, poly-N-acetyl-glucosamine, by elevating icaADBC mRNA levels. Unexpectedly, c-di-GMP synthesis was found to be irrelevant for the ability of GdpS to elevate icaADBC expression. Mutagenesis of the GGEEF motif essential for diguanylate cyclase activity did not impair GdpS, and the N-terminal fragment of GdpS lacking the GGDEF domain partially complemented the gdpS mutation. Furthermore, heterologous diguanylate cyclases expressed in trans failed to complement the gdpS mutation, and the purified GGDEF domain from GdpS possessed no diguanylate cyclase activity in vitro. The gdpS gene from Staphylococcus aureus exhibited similar characteristics to its S. epidermidis ortholog, suggesting that the GdpS-mediated signal transduction is conserved in staphylococci. Therefore, GdpS affects biofilm formation through a novel c-di-GMP-independent mechanism involving increased icaADBC mRNA levels and exopolysaccharide biosynthesis. Our data raise the possibility that staphylococci cannot synthesize c-di-GMP and have only remnants of a c-di-GMP signaling pathway.

joint and a coding joint (Roth et al., 1992a,b; Schlissel TX 77030, USA et al., 1993;Ramsden and Gellert, 1995; Zhu and Roth, 2 Corresponding author 1995). Experiments using cell-free systems have established that RAG-1 and RAG-2, the products of the In V(D)J recombination, double-strand breaks (DSBs) recombination activating genes (Schatz et al., 1989; are introduced at recombination signal sequences Oettinger et al., 1990), are the only protein factors (RSSs) which consist of three distinct elements: a necessary for proper DSB formation at an RSS (McBlane heptamer, a 12 or 23 nucleotide spacer and a nonamer . et al., 1995). Efficient DSB formation requires a 12/23 RSS pair andThe precise roles of the components of the RSS in the occurs at both RSS in a temporally coupled fashion cleavage reaction remain undefined. Mutational analysis (coupled cleavage). It remains unknown which RSS has shown that the heptamer, spacer and nonamer elements elements are important for coupled cleavage. Furtherare all important for efficient recombination in vivo (Hesse more, it has not been established whether some RSS et al., 1989;Akamatsu et al., 1994). While these studies components are critical only for cleavage in cis, with provided important information about the requirements others mainly promoting cleavage in trans at the for recombination under physiological conditions, the partner RSS. We investigated these questions by anaexperimental systems used measured only completed lyzing the effects of RSS mutations on the formation recombination events and could not assess effects on the of DSBs in vivo. The abundance of DSBs in cis (at the individual cleavage and joining steps. mutant RSS) and in trans (at the consensus RSS) wasMore recently, the cleavage step has been studied determined using an established ligation-mediated PCR in cell-free systems. The behavior of this reaction is assay. We also developed a Southern blotting approach dramatically affected by the incubation conditions. In the that allows the first direct measurement of dual and presence of Mg 2ϩ , efficient cleavage requires a 12/23 RSS single RSS cleavage in vivo. Our results demonstrate pair and generally occurs at both RSSs in a temporally that the heptamer, spacer and nonamer elements are coupled fashion (termed coupled cleavage) (Eastman et al., all required for coupled cleavage in vivo. These studies van Gent et al., 1996). These data suggest that also provide evidence for cleavage events involving a under these reaction conditions DSB formation may occur single RSS both in mutant substrates and in substrates in the context of a synaptic complex involving a 12/23 containing a consensus 12/23 RSS pair.RSS pair, an interpretation that is supported by analysis Keywords: double-strand break/RAG/recombination of several substrates bearing RSS mutations (Eastman signal sequence/synapsis/V(D) J recombination et al., 1996). However, in the presence of Mn 2ϩ , cleavage at the two RSSs is uncoupled, and substrates containing a single RSS are cleaved eff...

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In the facultatively phototrophic proteobacterium Rhodobacter sphaeroides, formation of the photosynthetic apparatus is oxygen dependent. When oxygen tension decreases, the response regulator PrrA of the global two-component PrrBA system is believed to directly activate transcription of the puf, puh, and puc operons, encoding structural proteins of the photosynthetic complexes, and to indirectly upregulate the photopigment biosynthesis genes bch and crt. Decreased oxygen also results in inactivation of the photosynthesis-specific repressor PpsR, bringing about derepression of the puc, bch, and crt operons. We uncovered a hierarchical relationship between these two regulatory systems, earlier thought to function independently. We also more accurately assessed the spectrum of gene targets of the PrrBA system. First, expression of the appA gene, encoding the PpsR antirepressor, is PrrA dependent, which establishes one level of hierarchical dominance of the PrrBA system over AppA-PpsR. Second, restoration of the appA transcript to the wild-type level is insufficient for rescuing phototrophic growth impairment of the prrA mutant, whereas inactivation of ppsR is sufficient. This suggests that in addition to controlling appA transcription, PrrA affects the activity of the AppA-PpsR system via an as yet unidentified mechanism(s). Third, PrrA directly activates several bch and crt genes, traditionally considered to be the PpsR targets. Therefore, in R. sphaeroides, the global PrrBA system regulates photosynthesis gene expression (i) by rigorous control over the photosynthesis-specific AppA-PpsR regulatory system and (ii) by extensive direct transcription activation of genes encoding structural proteins of photosynthetic complexes as well as genes encoding photopigment biosynthesis enzymes.In the facultatively phototrophic alphaproteobacterium Rhodobacter sphaeroides, photosynthesis (PS) operates under anoxic conditions. A decrease in oxygen tension triggers significant upregulation of PS gene transcription (35,38). The photosynthetic apparatus is comprised of the reaction center, encoded by the puh and puf operons, and two light-harvesting complexes, encoded by the puf and puc operons. Enzymes involved in the biosynthesis of photosynthetic pigments, i.e., bacteriochlorophyll a and carotenoids, are encoded by the bch and crt genes, respectively. Most PS-specific genes are located in the R. sphaeroides PS gene cluster, whereas the puc operons are located separately (4, 49). Three major regulatory systems control oxygen-dependent transcription of PS genes. One of these is composed of the antirepressor AppA (15, 17) and the repressor PpsR (7, 36) and is primarily responsible for the regulation of PS genes (30). Two other systems are global regulatory systems, i.e., the redox-responsive two-component system PrrBA (2, 47) and the anaerobic activator FnrL (48).

Background: V(D)J recombination is initiated by the introduction of double-stranded breaks (DSB) adjacent to recombination signal sequences (RSS). Each RSS contains a conserved heptamer and a conserved nonamer element separated by a 12 or 23 nucleotide spacer. In vivo, efficient recombination requires one RSS of each spacer length, although it has been unclear whether this '12/23 rule' regulates cleavage, joining, or both.