In Escherichia coli, the dinB gene is required for the SOS-induced lambda untargeted mutagenesis pathway and confers a mutator phenotype to the cell when the gene product is overexpressed. Here, we report that the purified DinB protein is a DNA polymerase. This novel E. coli DNA polymerase (pol IV) is shown to be strictly distributive, devoid of proofreading activity, and prone to elongate bulged (misaligned) primer/template structures. Site-directed mutagenesis experiments of dinB also demonstrate that the polymerase activity of DinB is required for its in vivo mutagenicity. Along with the sequence homologies previously found within the UmuC-like protein family, these results indicate that the uncovered DNA polymerase activity may be a common feature of all these homologous proteins.

ABSTRACTdinP is an Escherichia coli gene recently identified at 5.5 min of the genetic map, whose product shows a similarity in amino acid sequence to the E. coli UmuC protein involved in DNA damage-induced mutagenesis. In this paper we show that the gene is identical to dinB, an SOS gene previously localized near the lac locus at 8 min, the function of which was shown to be required for mutagenesis of nonirradiated phage infecting UV-preirradiated bacterial cells (termed UTM for untargeted mutagenesis). A newly constructed dinP null mutant exhibited the same defect for UTM as observed previously with a dinB::Mu mutant, and the defect was complemented by plasmids carrying dinP as the only intact bacterial gene. Furthermore, merely increasing the dinP gene expression, without UV irradiation or any other DNAdamaging treatment, resulted in a strong enhancement of mutagenesis in Flac plasmids; at most, 800-fold increase in the G6-to-G5 change. The enhanced mutagenesis did not depend on recA, uvrA, or umuDC. Thus, our results establish that E. coli has at least two distinct pathways for SOS-induced mutagenesis: one dependent on umuDC and the other on dinB͞P.

The hOGG1 gene encodes a DNA glycosylase that excises 8-hydroxyguanine (oh 8 Gua) from damaged DNA. Structural analyses of the hOGG1 gene and its transcripts were performed in normal and lung cancer cells. Due to a genetic polymorphism at codon 326, hOGG1-Ser 326 and hOGG1-Cys 326 proteins were produced in human cells. Activity in the repair of oh 8 Gua was greater in hOGG1-Ser 326 protein than in hOGG1-Cys 326 protein in the complementation assay of an E. coli mutant defective in the repair of oh 8 Gua. Two isoforms of hOGG1 transcripts produced by alternative splicing encoded distinct hOGG1 proteins: one with and the other without a putative nuclear localization signal. Loss of heterozygosity at the hOGG1 locus was frequently (15/ 23, 62.2%) detected in lung cancer cells, and a cell line NCI-H526 had a mutation leading to the formation of the transcripts encoding a truncated hOGG1 protein. However, the oh 8 Gua levels in nuclear DNA were similar among lung cancer cells and leukocytes irrespective of the type of hOGG1 proteins expressed. These results suggest that the oh 8 Gua levels are maintained at a steady level, even though multiple hOGG1 proteins are produced due to genetic polymorphisms, mutations and alternative splicing of the hOGG1 gene.

Haplotype *3 harboring a nonsynonymous SNP, 208G>A (Ala70Thr), decreased clearance of gemcitabine, and increased incidences of neutropenia when patients were coadministered platinum-containing drugs or fluorouracil.

The entire nucleotide sequence of the pil region of the IncI1 plasmid R64 was determined. Analysis of the sequence indicated that 14 genes, designated pilI through pilV, are involved in the formation of the R64 thin pilus. Protein products of eight pil genes were identified by the maxicell procedure. The pilN product was shown to be a lipoprotein by an experiment using globomycin. A computer search revealed that several R64 pil genes have amino acid sequence homology with proteins involved in type IV pilus biogenesis, protein secretion, and transformation competence. The pilS and pilV products were suggested to be prepilins for the R64 thin pilus, and the pilU product appears to be a prepilin peptidase. These results suggest that the R64 thin pilus belongs to the type IV family, specifically group IVB, of pili. The requirement of the pilR and pilU genes for R64 liquid mating was demonstrated by constructing their frameshift mutations. Comparison of three type IVB pilus biogenesis systems, the pil system of R64, the toxin-coregulated pilus (tcp) system of Vibrio cholerae, and the bundle-forming pilus (bfp) system of enteropathogenic Escherichia coli, suggests that they have evolved from a common ancestral gene system.Type IV pili are rod-like surface appendages produced by gram-negative bacteria such as Pseudomonas aeruginosa, Neisseria gonorrhoeae, Moraxella bovis, Myxococcus xanthus, and Vibrio cholerae, as well as enteropathogenic and enterotoxigenic Escherichia coli (for reviews, see references 27, 28, and 35). They are flexible, with a diameter of 6 to 7 nm and a length of up to 20 m. They are produced at the polar position of the bacterial cell. Many type IV pili play important roles in the attachment of bacterial pathogens to membranes of eukaryotic host cells, as do the other pili (13, 35). Type IV pili are also associated with the twitching motility of various bacteria and with the social motility of myxobacteria (40).Type IV pili are composed of pilin subunits (35). Pilin molecules from various bacteria have amino acid sequence homology (see Fig. 4E). The type IV pilin family is usually divided into two groups. Group A consists of pilins from P. aeruginosa, N. gonorrhoeae, M. bovis, and so on. They are closely related in amino acid sequence and are produced from prepilin molecules through the cleavage of 6-to 7-amino-acid signal peptides. The N-terminal amino acid of type IVA mature pilins is phenylalanine and is N methylated. Group B pilins, including toxin-coregulated pilus (tcp) in V. cholerae (26) and bundleforming pilus (bfp) in enteropathogenic E. coli (33,34), are substantively different from type IVA pilins. Their signal peptides are longer than those of type IVA pilins. The N-terminal amino acid of type IVB mature pilins is methionine or leucine.The C-terminal amino acid (glycine) of signal peptides and the 5th amino acid (glutamic acid) of mature pilins are completely conserved among type IVA and type IVB prepilins and related proteins (see Fig. 4E). A long hydrophobic segment is present at th...

We report the cloning of a human homolog of the yeast OGGC1 gene, which encodes a DNA glycosylase that excises an oxidatively damaged form of guanine, 8-hydroxyguanine (also known as 7,8-dihydro-8-oxoguanine). Since the deduced amino acid sequence (68 amino acids) of a human expressed sequence tag, N55394, matched a short stretch of yeast OGG1 protein with greater than 40% amino acid identity, a full length cDNA clone was isolated from a HeLa cell cDNA library with the N55394 clone as a probe. The cDNA clone encodes a predicted protein of 345 amino acids which is homologous to yeast OGG1 protein throughout the entire polypeptide sequence and shares 38% amino acid identity with yeast OGG1 protein. Moreover, we found that both a human homolog and yeast OGG1 protein possess two distinct DNA binding motifs, a helix-hairpin-helix (HhH) motif and a C2H2 zinc finger like motif, and a domain homologous to human and E. coli MutY proteins. Expression of a human homolog suppressed spontaneous mutagenesis of an E. coli (mutM mutY) mutant as in the case of yeast OGG1 protein. The gene was ubiquitously expressed in a variety of human organs and mapped to chromosome 3p26.2. These results strongly suggest that the gene isolated here is a human counterpart of the yeast OGGI gene and is involved in the repair of oxidative DNA damage in human cells.

To locate the transfer region of the 122-kiloase plasmid R64drd-11 belonging to incompatibility group I1, a series of deletion derivatives was constructed by in vitro recombinant DNA techniques followed by double homologous recombination in vivo. A plasmid designated pKK609 and bearing a 56.7-kilobase R64 sequence was the smallest transferable plasmid. A plasmid designated pKK610 and no longer possessing the 44-base-pair sequence from one end of pKK609 essential for the R64 onT function failed to transfer, indicating that the oriT sequence of the R64 transfer system is located at one end. The other end of the R64 transfer region comprises a DNA segment of about 19 kilobases responsible for pilus formation. Shufflon, DNA with a novel rearrangement in R64, was found to be involved in pilus formation.Bacterial conjugation is a complex process in which plasmid DNA is transferred from donor to recipient cells by a mechanism requiring cell-to-cell contact (for reviews, see references 10 and 25). The transfer system of IncF plasmids has been extensively studied. The transfer region of an IncF plasmid consists of more than 25 genes organized in a single contiguous region spanning 33 kilobases (kb), whose products are involved in pilus formation, stabilization of mating pairs, conjugative DNA metabolism, and surface exclusion.Incll plasmids such as R64, ColIb, and R144 also transfer by a mechanism similar to that of IncF plasmids. Despite its general similarity to the IncF conjugation system, however, the IncI transfer system exhibits three distinctive features. (i) Incl plasmids form two types of sex pili, one thick and the other thin, both morphologically and antigenically distinct from the IncF plasmid pilus (1, 2). (ii) Incl plasmids carry the sog gene responsible for suppressing dnaG mutations in Escherichia coli (7,18). The sog gene encodes DNA primase. (iii) An Incl plasmid shows a complex DNA rearrangement mediated by a unique structure designated shufflon (12-14). R64 shufflon consists of four DNA segments flanked and separated by seven 19-base-pair (bp) repeat sequences. The site-specific recombination between any two inverted repeats results in the inversion of DNA segments independently or in groups. The recombination is mediated by the gene rci, which is located adjacent to shufflon (17). Shufflon may function as a biological switch for the selection of one of seven genes in which the N-terminal three-quarters are constant and the C-terminal quarter is variable (14). The gene with the changing C-terminal region is tentatively named pilV in this paper. The C-terminal portions of the seven genes are called A, A', B, B', C, C', and D (14).Recently, we cloned, mapped, and sequenced the oriT region of the R64drd-JJ plasmid (15 system have been clarified, the global organization of IncIl plasmid transfer genes has yet to be determined in detail.The physical maps of R64, ColIb, and R144 are presently available (7,9,22,24). Recently, the organization of Collb- P9 transfer genes was analyzed by transposon mutagenesis...

In order to identify single nucleotide polymorphisms (SNPs) and haplotype frequencies of CYP3A5 in a Japanese population, we sequenced the proximal promoter region, all exons, and the surrounding intronic regions using genomic DNA from 187 Japanese subjects. Thirteen SNPs, including seven novel ones: 13108T>C, 16025A>G, 16903A>G, 16993C>G, 27448C>A, 29782A>G, and 31551T>C (A of the translational start codon of GenBank Accession # NG_000004.2 is numbered "1" according to the CYP Allele Nomenclature), were identified. The most common SNP was 6986A>G (key SNP for CYP3A5*3), with a 0.759 frequency. Two novel SNPs, 29782A>G (I456V) and 31551T>C (I488T), as well as 12952T>C (*5 marker) were found, but these alterations were always associated with the *3A marker SNPs, 6986A>G and 31611C>T. Using these 13 SNPs, haplotype analysis was performed and five novel *1 haplotypes (subtypes) (*1e to *1i) and six novel *3 haplotypes (subtypes) (*3d to *3i) were identified. Our findings suggest that CYP3A5*3 is the major defective allele and that other functional exonic SNPs are rare in the Japanese.