A Green Nonsulfur Bacterium, Dehalococcoides ethenogenes, with the LexA Binding Sequence Found in Gram-Positive Organisms

Dehalococcoides ethenogenes is a member of the physiologically diverse division of green nonsulfur bacteria. Using a TBLASTN search, the D. ethenogenes lexA gene has been identified, cloned, and expressed and its protein has been purified. Mobility shift assays revealed that the D. ethenogenes LexA protein specifically binds to both its own promoter and that of the uvrA gene, but not to the recA promoter. Our results demonstrate that the D. ethenogenes LexA binding site is GAACN(4)GTTC, which is identical to that found in gram-positive bacteria. In agreement with this fact, the Bacillus subtilis DinR protein binds specifically to the D. ethenogenes LexA operator. This constitutes the first non-gram-positive bacterium exhibiting a LexA binding site identical to that of B. subtilis

Geobacter sulfurreducens Has Two Autoregulated lexA Genes Whose Products Do Not Bind the recA Promoter: Differing Responses of lexA and recA to DNA Damage

The Escherichia coli LexA protein was used as a query sequence in TBLASTN searches to identify the lexA gene of the δ-proteobacterium Geobacter sulfurreducens from its genome sequence. The results of the search indicated that G. sulfurreducens has two independent lexA genes designated lexA1 and lexA2. A copy of a dinB gene homologue, which in E. coli encodes DNA polymerase IV, is present downstream of each lexA gene. Reverse transcription-PCR analyses demonstrated that, in both cases, lexA and dinB constitute a single transcriptional unit. Electrophoretic mobility shift assays with purified LexA1 and LexA2 proteins have shown that both proteins bind the imperfect palindrome GGTTN(2)CN(4)GN(3)ACC found in the promoter region of both lexA1 and lexA2. This sequence is also present upstream of the Geobacter metallireducens lexA gene, indicating that it is the LexA box of this bacterial genus. This palindrome is not found upstream of either the G. sulfurreducens or the G. metallireducens recA genes. Furthermore, DNA damage induces expression of the lexA-dinB transcriptional unit but not that of the recA gene. However, the basal level of recA gene expression is dramatically higher than that of the lexA gene. Likewise, the promoters of the G. sulfurreducens recN, ruvAB, ssb, umuDC, uvrA, and uvrB genes do not contain the LexA box and are not likely to bind to the LexA1 or LexA2 proteins. G. sulfurreducens is the first bacterial species harboring a lexA gene for which a constitutive expression of its recA gene has been described

Characterization of a New LexA Binding Motif in the Marine Magnetotactic Bacterium Strain MC-1

MC-1 is a marine, magnetotactic bacterium that is phylogenetically associated with the alpha subclass of the Proteobacteria and is the first and only magnetotactic coccus isolated in pure culture to date. By using a TBLASTN search, a lexA gene was identified in the published genome of MC-1; it was subsequently cloned, and the protein was purified to >90% purity. Results from reverse transcription-PCR analysis revealed that the MC-1 lexA gene comprises a single transcriptional unit with two open reading frames encoding proteins of unknown function and with a rumA-like gene, a homologue of the Escherichia coli umuD gene. Mobility shift assays revealed that this LexA protein specifically binds both to its own promoter and to that of the umuDC operon. However, MC-1 LexA does not bind to the promoter regions of other genes, such as recA and uvrA, that have been previously reported to be regulated by LexA in bacterial species belonging to the alpha subclass of the Proteobacteria. Site-directed mutagenesis of both the lexA and umuDC operator regions demonstrated that the sequence CCTN(10)AGG is the specific target motif for the MC-1 LexA protein

Controlling the subcellular localization of DNA polymerases ι and η via interactions with ubiquitin

Y‐family DNA polymerases have spacious active sites that can accommodate a wide variety of geometric distortions. As a consequence, they are considerably more error‐prone than high‐fidelity replicases. It is hardly surprising, therefore, that the in vivo activity of these polymerases is tightly regulated, so as to minimize their inadvertent access to primer‐termini. We report here that one such mechanism employed by human cells relies on a specific and direct interaction between DNA polymerases ι and η with ubiquitin (Ub). Indeed, we show that both polymerases interact noncovalently with free polyUb chains, as well as mono‐ubiquitinated proliferating cell nuclear antigen (Ub‐PCNA). Mutants of polι (P692R) and polη (H654A) were isolated that are defective in their interactions with polyUb and Ub‐PCNA, whilst retaining their ability to interact with unmodified PCNA. Interestingly, the polymerase mutants exhibit significantly lower levels of replication foci in response to DNA damage, thereby highlighting the biological importance of the polymerase–Ub interaction in regulating the access of the TLS polymerases to stalled replication forks in vivo.This work was supported by funds from the NICHD/NIH Intramural Research Program. We thank Alan Tomkinson and Sangeetha Vijayakumar for the gift of purified human PCNA; Michael Matunis for kindly providing the SUMO‐1 cDNA; Kathleen Downey for the cDNA to POLD1; Vladimir Podust for cDNA to POLD3; Mike O'Donnell for the yeast RFC expression plasmid; Alan Lehmann for pEGFP‐polη H654A; and Ivan Dikic, Alan Lehman, Peter Burgers and Bradley Wouters for kindly sharing data prior to publication. Microscopy Imaging was performed at the Microscopy & Imaging Core Facility (NICHD/NIH) with the assistance of Dr Vincent Schram.Peer reviewe

JOURNAL OF BACTERIOLOGY, Nov. 2002, p. 6073--6080 Vol. 184, No. 21 0021-9193/02/$04.000 DOI: 10.1128/JB.184.21.6073--6080.2002

this report, there were no data available pertaining to the motif recognized by LexA in this bacterial division. Finally, * Corresponding author. Mailing address: Universitat Autonoma de Barcelona, Departament de Genetica i Microbiologia, Unitat de Microbiologia, Edifici C, Bellaterra, 08193 Barcelona, Spain. Phone: 34 93 581 1837. Fax: 34 93 581 23 87. E-mail for Antonio R. Fernandez de Henestrosa: [email protected]. E-mail for Jordi Barbe: [email protected]

Natural Retinol Analogs Potentiate the Effects of Retinal on Aged and Photodamaged Skin: Results from In Vitro to Clinical Studies

Abstract Introduction Plants are a source of natural ingredients with retinol-like properties that can deliver anti-aging benefits without the side effects typically associated with retinoid use. We hypothesized that by combining two such analogs, bakuchiol (BAK) and Vigna aconitifolia extract (VAE), with the potent retinoid retinal (RAL), the anti-photoaging potential of RAL could be enhanced without compromising its skin irritation profile. The purpose of this study was to demonstrate that BAK and VAE potentiate the anti-photoaging activity of RAL. Methods Gene expression profiling of full-thickness reconstructed skin was first used to examine the impact of BAK or VAE in combination with RAL on skin biology. Next, the irritative potential of this combination, and its capacity to reverse key signs of photoaging in an ex vivo model was assessed. Finally, a proof-of-concept open label clinical study was performed to evaluate the anti-photoaging capacity and skin compatibility of a cosmetic formulation (tri-retinoid complex; 3RC) containing this complex in combination with other well characterized anti-photoaging ingredients. Results In vitro profiling suggested that combining 0.1% RAL with BAK or VAE potentiates the effect of RAL on keratinocyte differentiation and skin barrier function without affecting its skin irritation profile. When formulated with other anti-photoaging ingredients, such as niacinamide and melatonin, 3RC reversed ultraviolet radiation-induced deficits in structural components of the dermal extracellular matrix, including hyaluronic acid and collagen. In vivo, it led to a reversal of clinical signs of age and photodamage, with statistically significant improvement to skin firmness (+5.6%), skin elasticity (+13.9%), wrinkle count (−43.2%), and skin tone homogeneity (+7.0%), observed within 28 days of once nightly use. Notably, the number of crow’s feet wrinkles was reduced in 100% of subjects. Furthermore, 3RC was very well tolerated. Conclusion These data suggest that 3RC is a highly effective and well-tolerated treatment for photoaging

Lack of sugar discrimination by human Pol µ requires a single glycine residue

DNA polymerase mu (Pol µ) is a novel family X DNA polymerase that has been suggested to play a role in micro-homology mediated joining and repair of double strand breaks. We show here that human Pol µ is not able to discriminate against the 2′-OH group of the sugar moiety. It inserts rNTPs with an efficiency that is <10-fold lower than that of dNTPs, in sharp contrast with the >1000-fold discrimination characteristic of most DNA-dependent DNA polymerases. The lack of sugar discrimination by Pol µ is demonstrated by its ability to add rNTPs to both DNA and RNA primer strands, and to insert both deoxy- and ribonucleotides on growing nucleic acid chains. 3D-modelling of human Pol µ based on the available Pol β and TdT structural information allowed us to predict candidate residues involved in sugar discrimination. Thus, a single amino acid substitution in which Gly433 residue of Pol µ was mutated to the consensus tyrosine present in Pol β, produced a strong increase in the discrimination against ribonucleotides. The unusual capacity to insert both rNTPs and dNTPs will be discussed in the context of the predicted roles of Pol µ in DNA repair