Cockayne syndrome group B protein is engaged in processing of DNA adducts of lipid peroxidation product trans-4-hydroxy-2-nonenal.

Cockayne syndrome complementation group B (CSB) protein is engaged in transcription-coupled repair (TCR) of UV induced DNA damage and its deficiency leads to progressive multisystem degeneration and premature aging. Here, we show that human CSB-deficient cells are hypersensitive to physiological concentrations (1-10 microM) of a lipid peroxidation product, trans-4-hydroxy-2-nonenal (HNE), and in response to HNE they develop a higher level of sister chromatid exchanges (SCEs) in comparison to the wild-type cells. HNE-DNA adducts block in vitro transcription by T7 RNA polymerase, as well as by HeLa cell-free extracts. Treatment of wild-type cells with 1-20 microM HNE causes dephosphorylation of the CSB protein, which stimulates its ATPase activity necessary for TCR. However, high HNE concentrations (100-200 microM) inhibit in vitro CSB ATPase activity as well as the transcription machinery in HeLa cell-free extracts. Cell lines expressing CSB protein mutated in different ATPase domains exhibit different sensitivities to HNE. The motif II mutant, which binds ATP, but is defective in ATP hydrolysis was as sensitive to HNE as CSB-null cells. In contrast, motif V mutant cells were as sensitive to HNE as were the cells bearing wild-type protein, while motif VI mutant cells showed intermediate sensitivity to HNE. These mutants exhibit decreased ATP binding, but retain residual ATPase activity. Homology modeling suggested that amino acids mutated in motifs II and VI are localized closer to the ATP binding site than amino acids mutated in ATPase motif V. These results suggest that HNE-DNA adducts are extremely toxic endogenous DNA lesion, and that their processing involves CSB. When these lesions are not removed from the transcribed DNA strand due to CSB gene mutation or CSB protein inactivation by high, pathological HNE concentrations, they may contribute to accelerated aging.

Structural basis of the sulphate starvation response in E. coli: crystal structure and mutational analysis of the cofactor-binding domain of the Cbl transcriptional regulator.

Cbl is a member of the large family of LysR-type transcriptional regulators (LTTRs) common in bacteria and found also in Archaea and algal chloroplasts. The function of Cbl is required in Escherichia coli for expression of sulphate starvation-inducible (ssi) genes, associated with the biosynthesis of cysteine from organic sulphur sources (sulphonates). Here, we report the crystal structure of the cofactor-binding domain of Cbl (c-Cbl) from E. coli. The overall fold of c-Cbl is very similar to the regulatory domain (RD) of another LysR family member, CysB. The RD is composed of two subdomains enclosing a cavity, which is expected to bind effector molecules. We have constructed and analysed several full-length Cbl variants bearing single residue substitutions in the RD that affect cofactor responses. Using in vivo and in vitro transcription assays, we demonstrate that pssuE, a Cbl responsive promoter, is down-regulated not only by the cofactor, adenosine phosphosulphate (APS), but also by thiosulphate, and, that the same RD determinants are important for the response to both cofactors. We also demonstrate the effects of selected site-directed mutations on Cbl oligomerization and discuss these in the context of the structure. Based on the crystal structure and molecular modelling, we propose a model for the interaction of Cbl with adenosine phosphosulphate.

Full-length cDNAs from chicken bursal lymphocytes to facilitate gene function analysis.

A large number of cDNA inserts were sequenced from a high-quality library of chicken bursal lymphocyte cDNAs. Comparisons to public gene databases indicate that the cDNA collection represents more than 2,000 new, full-length transcripts. This resource defines the structure and the coding potential of a large fraction of B-cell specific and housekeeping genes whose function can be analyzed by disruption in the chicken DT40 B-cell line.

Identification of new genes regulated by the Crt1 transcription factor, an effector of the DNA damage checkpoint pathway in Saccharomyces cerevisiae.

The Crt1 (RFX1) protein in Saccharomyces cerevisiae is an effector of the DNA damage checkpoint pathway. It recognizes a 13-bp cis-regulatory element in the 5'-untranslated region (5'-UTR) of the ribonucleotide reductase genes RNR2, RNR3, and RNR4; the HUG1 gene; and itself. We calculated the weight matrix representing the Crt1p binding site motif according to analysis of the 5'-UTR sequences of the genes that are under its regulation. We subsequently searched the 5'-UTR sequences of all the genes in the yeast genome for the occurrence of this motif. The motif was found in regulatory regions of 30 genes. A statistical analysis showed that it is unlikely that a random gene cluster contains the motif conserved as well as the Crt1p binding site. Analysis of microarray data provided supporting evidence for five putative Crt1p targets: FSH3, YLR345W, UBC5, NDE2, and NTH2. We used reverse transcription-PCR to compare the expression levels of these genes in wild-type and crt1Delta strains. Our results indicated that FSH3, YLR345W, and NTH2 are indeed under the regulation of Crt1p. Sequence analysis of the FSH3p indicated that this protein may be involved in folate metabolism either by carrying serine hydrolase activity required for the novel metabolic pathway involving dihydrofolate reductase (DHFR) or by directly interacting with the DHFR enzyme. We postulate that Crt1p may influence deoxyribonucleotide synthesis not only by regulating expression of the RNR genes but also by modulating DHFR activity. FSH3p shares significant sequence similarity with the product of the human tumor suppressor gene OVCA2. YLR345Wp and NTH2p are enzymes involved in the central metabolism under stress conditions.

Identification of activating region (AR) of Escherichia coli LysR-type transcription factor CysB and CysB contact site on RNA polymerase alpha subunit at the cysP promoter.

CysB is a LysR-type transcriptional regulator (LTTR) controlling the expression of numerous genes involved in bacterial sulphur assimilation via cysteine biosynthesis. Our previous mutational analysis of CysB identified several residues within the N-terminal domain crucial for DNA-binding function. Here, we focus on the functional significance of CysB residues localized in the turn between the alpha2 and alpha3 helices forming an N-terminal helix-turn-helix motif. On the basis of the characteristics of alanine-substituted mutants, we propose that CysB residues Y27, T28 and S29, lying in this turn region, comprise an 'activating region' (AR) that is crucial for positive control of the cysP promoter, but not for DNA binding and inducer response activities of CysB. Using a library of alanine substitutions in the C-terminal domain of the RNAP alpha subunit (alpha-CTD), we identify several residues in alpha-CTD that are important for CysB-dependent transcription from the cysP promoter. After probing potential protein-protein contacts in vivo with a LexA-based two-hybrid system, we propose that the '273 determinant' on alpha-CTD, including residues K271 and E273, represents a target for interaction with CysB at the cysP promoter.

The structure of full-length LysR-type transcriptional regulators. Modeling of the full-length OxyR transcription factor dimer.

The LysR-type transcriptional regulators (LTTRs) comprise the largest family of prokaryotic transcription factors. These proteins are composed of an N-terminal DNA binding domain (DBD) and a C-terminal cofactor binding domain. To date, no structure of the DBD has been solved. According to the SUPERFAMILY and MODBASE databases, a reliable homology model of LTTR DBDs may be built using the structure of the Escherichia coli ModE transcription factor, containing a winged helix- turn-helix (HTH) motif, as a template. The remote, but statistically significant, sequence similarity between ModE and LTTR DBDs and an alignment generated using SUPERFAMILY and MODBASE methods was independently confirmed by alignment of sequence profiles representing ModE and LTTR family DBDs. Using the crystal structure of the E.coli OxyR C-terminal domain and the DBD alignments we constructed a structural model of the full-length dimer of this LTTR family member and used it to investigate the mode of protein-DNA interaction. We also applied the model to interpret, in a structural context, the results of numerous biochemical studies of mutated LTTRs. A comparison of the LTTR DBD model with the structures of other HTH proteins also provides insights into the interaction of LTTRs with the C-terminal domain of the RNA polymerase alpha subunit.