Tomoo Ogi

Identification, Timing, and Signal Specificity of Pseudomonas aeruginosa Quorum-Controlled Genes: a Transcriptome Analysis

There are two interrelated acyl-homoserine lactone quorum-sensing-signaling systems in Pseudomonas aeruginosa. These systems, the LasR-LasI system and the RhlR-RhlI system, are global regulators of gene expression. We performed a transcriptome analysis to identify quorum-sensing-controlled genes and to better understand quorum-sensing control of P. aeruginosa gene expression. We compared gene expression in a LasI-RhlI signal mutant grown with added signals to gene expression without added signals, and we compared a LasR-RhlR signal receptor mutant to its parent. In all, we identified 315 quorum-induced and 38 quorum-repressed genes, representing about 6% of the P. aeruginosa genome. The quorum-repressed genes were activated in the stationary phase in quorum-sensing mutants but were not activated in the parent strain. The analysis of quorum-induced genes suggests that the signal specificities are on a continuum and that the timing of gene expression is on a continuum (some genes are induced early in growth, most genes are induced at the transition from the logarithmic phase to the stationary phase, and some genes are induced during the stationary phase). In general, timing was not related to signal concentration. We suggest that the level of the signal receptor, LasR, is a critical trigger for quorum-activated gene expression. Acyl-homoserine lactone quorum sensing appears to be a system that allows ordered expression of hundreds of genes during P. aeruginosa growth in culture.

Identification of additional genes belonging to the LexA regulon in Escherichia coli

Exposure of Escherichia coli to a variety of DNA‐damaging agents results in the induction of the global ‘SOS response’. Expression of many of the genes in the SOS regulon are controlled by the LexA protein. LexA acts as a transcriptional repressor of these unlinked genes by binding to specific sequences (LexA boxes) located within the promoter region of each LexA‐regulated gene. Alignment of 20 LexA binding sites found in the E. coli chromosome reveals a consensus of 5′‐TACTG(TA)5CAGTA‐3′. DNA sequences that exhibit a close match to the consensus are said to have a low heterology index and bind LexA tightly, whereas those that are more diverged have a high heterology index and are not expected to bind LexA. By using this heterology index, together with other search criteria, such as the location of the putative LexA box relative to a gene or to promoter elements, we have performed computational searches of the entire E. coli genome to identify novel LexA‐regulated genes. These searches identified a total of 69 potential LexA‐regulated genes/operons with a heterology index of < 15 and included all previously characterized LexA‐regulated genes. Probes were made to the remaining genes, and these were screened by Northern analysis for damage‐inducible gene expression in a wild‐type lexA+ cell, constitutive expression in a lexA(Def) cell and basal expression in a non‐inducible lexA(Ind−) cell. These experiments have allowed us to identify seven new LexA‐regulated genes, thus bringing the present number of genes in the E. coli LexA regulon to 31. The potential function of each newly identified LexA‐regulated gene is discussed.

Three DNA Polymerases, Recruited by Different Mechanisms, Carry Out NER Repair Synthesis in Human Cells

Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both pol kappa and pol delta, and both polymerases can be recovered in the same repair complexes. Pol kappa is recruited to repair sites by ubiquitinated PCNA and XRCC1 and pol delta by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on pol epsilon, recruitment of which is dependent on the alternative clamp loader CTF18-RFC.

Polκ protects mammalian cells against the lethal and mutagenic effects of benzo[a]pyrene

Several low-fidelity DNA polymerases have recently been discovered that are able to bypass DNA lesions during DNA synthesis in vitro. The efficiency and accuracy of lesion bypass is, however, both polymerase and lesion specific. For example, in vitro studies revealed that human DNA polymerase κ (Polκ) is unable to insert a base opposite a cis-syn thymine-thymine dimer or cisplatin adduct, yet can bypass some DNA lesions such as abasic site and acetylaminofluorene-adducted guanine in an error-prone manner. More importantly, Polκ is able to bypass benzo[a]pyrene (B[a]P)-adducted guanine accurately and efficiently. To investigate the biological function of Polκ, we have generated mouse embryonic stem (ES) cells deficient in the Polk gene encoding the enzyme. Polk-deficient ES cells grow normally and their sensitivities to UV and x-ray radiation are only slightly affected. In contrast, the mutant cells are highly sensitive to both killing and mutagenesis induced by B[a]P. Furthermore, the spectrum of mutations recovered in the Polk-deficient cells is different from that in the wild-type cells. Thus, our results indicate that Polκ plays an important role in suppressing mutations at DNA lesions generated by B[a]P.

The Y-family DNA polymerase κ (pol κ) functions in mammalian nucleotide-excision repair

DNA polymerase κ (pol κ) is a member of the Y-family of DNA polymerases that are thought to function in translesion synthesis (TLS) past different types of DNA damage. Here, we show that pol κ-deficient mouse cells have substantially reduced (but not absent) levels of nucleotide excision repair (NER) of UV damage, as measured by several methods. Our results provide evidence for an unexpected role for pol κ in mammalian NER.

Mutation enhancement by DINB1, a mammalian homologue of the Escherichia coli mutagenesis protein dinB.

The Escherichia coli dinB gene is an SOS gene known to be required for λ phage untargeted mutagenesis. When over‐expressed, it exhibits a potent mutagenic activity without any exogenous treatment to damage DNA. Frameshift mutations at a run of identical bases are most enhanced. The product DinB is structurally related to the E. coli UmuC protein and the Saccharomyces cerevisiae Rev1 and Rad30 proteins, all of which are shown to be involved in bypass synthesis at a DNA lesion.

Malfunction of nuclease ERCC1-XPF results in diverse clinical manifestations and causes Cockayne syndrome, xeroderma pigmentosum, and Fanconi anemia.

Cockayne syndrome (CS) is a genetic disorder characterized by developmental abnormalities and photodermatosis resulting from the lack of transcription-coupled nucleotide excision repair, which is responsible for the removal of photodamage from actively transcribed genes. To date, all identified causative mutations for CS have been in the two known CS-associated genes, ERCC8 (CSA) and ERCC6 (CSB). For the rare combined xeroderma pigmentosum (XP) and CS phenotype, all identified mutations are in three of the XP-associated genes, ERCC3 (XPB), ERCC2 (XPD), and ERCC5 (XPG). In a previous report, we identified several CS cases who did not have mutations in any of these genes. In this paper, we describe three CS individuals deficient in ERCC1 or ERCC4 (XPF). Remarkably, one of these individuals with XP complementation group F (XP-F) had clinical features of three different DNA-repair disorders--CS, XP, and Fanconi anemia (FA). Our results, together with those from Bogliolo et al., who describe XPF alterations resulting in FA alone, indicate a multifunctional role for XPF.

Collaborative Action of Brca1 and CtIP in Elimination of Covalent Modifications from Double-Strand Breaks to Facilitate Subsequent Break Repair

Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3′ and 5′ ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3′ single-strand overhang at “clean” DSBs, thus initiating homologous recombination (HR)–dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3′ single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIPS332A/−/− cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP+/−/− cells. Finally, CtIPS332A/−/−BRCA1−/− and CtIP+/−/−BRCA1−/− showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.

Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair

UV-sensitive syndrome (UVSS) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma. Despite mild clinical features, cells from individuals with UVSS, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER), which removes DNA damage in actively transcribed genes. Three of the seven known UVSS cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively). The remaining four individuals with UVSS, one of whom is described for the first time here, formed a separate UVSS-A complementation group; however, the responsible gene was unknown. Using exome sequencing, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UVSS-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER–deficient disorders.

Expression of human and mouse genes encoding polκ: testis-specific developmental regulation and AhR-dependent inducible transcription

Backgrounds Human polκ is a newly identified low‐fidelity DNA polymerase. While the enzyme bypasses an abasic site and acetylaminofluorene‐adduct in an error‐prone manner, it bypasses benzo[a]pyrene‐N2‐dG lesions in a mostly error‐free manner by incorporating predominantly dC opposite the bulky lesions. Benzo[a]pyrene (B[a]P) is activated through intracellular process mediated by the arylhydrocarbon receptor (AhR, also called the dioxin receptor), which is a ligand‐activated transcription factor with high affinities for aromatic compounds such as B[a]P and dioxin.