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R Woodgate

Publications and source records attributed to R Woodgate.

At least 37 records · Page 2Linked to original sources
PubMed2000

Misinsertion and bypass of thymine-thymine dimers by human DNA polymerase iota.

Human DNA polymerase iota (pol(iota)) is a recently discovered enzyme that exhibits extremely low fidelity on undamaged DNA templates. Here, we show that poliota is able to facilitate limited translesion replication of a thymine-thymine cyclobutane pyrimidine dimer (CPD). More importantly, however, the bypass event is highly erroneous. Gel kinetic assays reveal that pol(iota) misinserts T or G opposite the 3' T of the CPD approximately 1.5 times more frequently than the correct base, A. While pol(iota) is unable to extend the T.T mispair significantly, the G.T mispair is extended and the lesion completely bypassed, with the same efficiency as that of the correctly paired A. T base pair. By comparison, pol(iota) readily misinserts two bases opposite a 6-4 thymine-thymine pyrimidine-pyrimidone photoproduct (6-4PP), but complete lesion bypass is only a fraction of that observed with the CPD. Our data indicate, therefore, that poliota possesses the ability to insert nucleotides opposite UV photoproducts as well as to perform unassisted translesion replication that is likely to be highly mutagenic.

Base Pair Mismatch↗
PubMed2000

poliota, a remarkably error-prone human DNA polymerase.

The Saccharomyces cerevisiae RAD30 gene encodes DNA polymerase eta. Humans possess two Rad30 homologs. One (RAD30A/POLH) has previously been characterized and shown to be defective in humans with the Xeroderma pigmentosum variant phenotype. Here, we report experiments demonstrating that the second human homolog (RAD30B), also encodes a novel DNA polymerase that we designate poliota. poliota, is a distributive enzyme that is highly error-prone when replicating undamaged DNA. At template G or C, the average error frequency was approximately 1 x 10(-2). Our studies revealed, however, a striking asymmetry in misincorporation frequency at template A and T. For example, template A was replicated with the greatest accuracy, with misincorporation of G, A, or C occurring with a frequency of approximately 1 x 10(-4) to 2 x 10(-4). In dramatic contrast, most errors occurred at template T, where the misincorporation of G was, in fact, favored approximately 3:1 over the correct nucleotide, A, and misincorporation of T occurred at a frequency of approximately 6.7 x 10(-1). These findings demonstrate that poliota is one of the most error-prone eukaryotic polymerases reported to date and exhibits an unusual misincorporation spectrum in vitro.

Base Sequence↗
PubMed2000

Roles of E. coli DNA polymerases IV and V in lesion-targeted and untargeted SOS mutagenesis.

The expression of the Escherichia coli DNA polymerases pol V (UmuD'2C complex) and pol IV (DinB) increases in response to DNA damage. The induction of pol V is accompanied by a substantial increase in mutations targeted at DNA template lesions in a process called SOS-induced error-prone repair. Here we show that the common DNA template lesions, TT (6-4) photoproducts, TT cis-syn photodimers and abasic sites, are efficiently bypassed within 30 seconds by pol V in the presence of activated RecA protein (RecA*), single-stranded binding protein (SSB) and pol III's processivity beta,gamma-complex. There is no detectable bypass by either pol IV or pol III on this time scale. A mutagenic 'signature' for pol V is its incorporation of guanine opposite the 3'-thymine of a TT (6-4) photoproduct, in agreement with mutational spectra. In contrast, pol III and pol IV incorporate adenine almost exclusively. When copying undamaged DNA, pol V exhibits low fidelity with error rates of around 10(-3) to 10(-4), with pol IV being 5- to 10-fold more accurate. The effects of RecA protein on pol V, and beta,gamma-complex on pol IV, cause a 15,000- and 3,000-fold increase in DNA synthesis efficiency, respectively. However, both polymerases exhibit low processivity, adding 6 to 8 nucleotides before dissociating. Lesion bypass by pol V does not require beta,gamma-complex in the presence of non-hydrolysable ATPgammaS, indicating that an intact RecA filament may be required for translesion synthesis.

DNA Damage↗
PubMed2000

Visualization of two binding sites for the Escherichia coli UmuD'(2)C complex (DNA pol V) on RecA-ssDNA filaments.

The heterotrimeric UmuD'(2)C complex of Escherichia coli has recently been shown to possess intrinsic DNA polymerase activity (DNA pol V) that facilitates error-prone translesion DNA synthesis (SOS mutagenesis). When overexpressed in vivo, UmuD'(2)C also inhibits homologous recombination. In both activities, UmuD'(2)C interacts with RecA nucleoprotein filaments. To examine the biochemical and structural basis of these reactions, we have analyzed the ability of the UmuD'(2)C complex to bind to RecA-ssDNA filaments in vitro. As estimated by a gel retardation assay, binding saturates at a stoichiometry of approximately one complex per two RecA monomers. Visualized by cryo-electron microscopy under these conditions, UmuD'(2)C is seen to bind uniformly along the filaments, such that the complexes are completely submerged in the deep helical groove. This mode of binding would impede access to DNA in a RecA filament, thus explaining the ability of UmuD'(2)C to inhibit homologous recombination. At sub-saturating binding, the distribution of UmuD'(2)C complexes along RecA-ssDNA filaments was characterized by immuno-gold labelling with anti-UmuC antibodies. These data revealed preferential binding at filament ends (most likely, at one end). End-specific binding is consistent with genetic models whereby such binding positions the UmuD'(2)C complex (pol V) appropriately for its role in SOS mutagenesis.

Binding Sites↗
PubMed2000

Selective inhibition of RecA functions by the Hc1 nucleoid condensation protein from Chlamydia trachomatis.

During the normal biphasic life cycle of Chlamydia trachomatis, the histone-like protein Hc1 promotes the condensation of nucleoids in elementary bodies, it may also displace nucleoproteins, including repair functions from chromatin. Hc1 was found to effectively inhibit the recombination and repair of the weak binding RecA430 mutant protein from Escherichia coli, but had minimal effects on the parental RecA(+) protein. Expression of Hc1 was also found to inhibit the repair activities of the C. trachomatis RecA protein but not recombination. These results suggest that chlamydial RecA may have evolved mechanisms to minimize Hc1 competition for recombinational activities.

Chlamydia trachomatis↗
PubMed2000

Perceptions of the role of the school in providing information and support to adolescent children of women with breast cancer.

Little is known about the needs of adolescents of women with breast cancer. The purpose of this study was to describe the perceptions of adolescents (ages 12-20) about the role of the school in assisting them in dealing with the cancer experience. An exploratory, qualitative study was done to elicit detailed descriptions of adolescent's needs for information and support in response to their mother's breast cancer. A convenience sample of 31 adolescents of women in five illness phases participated in semi-structured interviews. In addition, two focus group interviews were conducted. Interviews were tape-recorded, transcribed and analysed using content analysis techniques. Findings specific to the adolescents' perceptions of the role of the school were discussed according to content, type, amount, timing, provider and quality of information. Support needs were discussed as type of support, source of support, amount, timing and focus. Although school personnel attempted to be supportive and adolescents received generic information about cancer, overall the needs of the adolescents were not adequately addressed. Recommendations for schools and health services in assisting adolescents to cope with this major life experience are made.

Adolescent↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

Intrinsic polymerase activities of UmuD'(2)C and MucA'(2)B are responsible for their different mutagenic properties during bypass of a T-T cis-syn cyclobutane dimer.

In wild-type Escherichia coli, translesion replication is largely dependent upon the UmuD'(2)C complex (DNA polymerase V [polV]) or its plasmid-encoded homologs, such as MucA'(2)B. Interestingly, both the efficiency of translesion replication of a T-T cis-syn dimer and the spectra of mutations observed are different in Umu- and Muc-expressing strains. We have investigated whether the polIII core is responsible for these differences by measuring the frequency of dimer bypass, the error rate of bypass, and the resulting mutation spectrum in mutants carrying a deletion of dnaQ (epsilon subunit) or holE (theta subunit) or carrying the dnaQ allele mutD5, which is deficient in proofreading but is competent in the structural function of epsilon, or the dnaE antimutator allele spq-2. The chromosomal copy of the umuDC operon was deleted in each strain, and the UmuDC, UmuD'C, MucAB, or MucA'B proteins were expressed from a low-copy-number plasmid. With only few exceptions, we found that the characteristically different mutation spectra resulting from Umu- and Muc-mediated bypass are maintained in all of the strains investigated, indicating that differences in the activity or structure of the polIII core are not responsible for the observed phenotype. We also demonstrate that the MucA'(2)B complex is more efficient in promoting translesion replication than the UmuD'(2)C proteins and show that, contrary to expectation, the T-T dimer is bypassed more accurately by MucA'(2)B than by UmuD'(2)C. These results are consistent with the view that in a wild-type cell, the polV-like enzymes are responsible for the spectra of mutations generated during translesion replication and that polIII may simply be required to fix the misincorporations as mutations by completing chromosomal replication. Our observations also show that the mutagenic properties of a lesion can depend strongly on the particular enzyme employed in bypass.

Bacterial Proteins↗
PubMed2000

Part I: an introduction to conducting qualitative research in children with cancer.

Over the past decade, pediatric nurse researchers have acknowledged the need to study children's cancer illness experiences within the qualitative research framework. Support for more qualitative research is based on the belief that it will afford researchers the opportunity to get closer to understanding children's perspectives of their cancer experience. A priori theories or generalizations by the researcher are not imposed; therefore, information emerging from the research is believed to be more a reflection of the perspectives of child participants and not adult researchers. Although pediatric oncology nurses may be interested in using more qualitative methods in their research, deciding on the appropriate qualitative research design may not always be so evident, considering that the adoption of qualitative inquiry in the study of childhood cancer is in its infancy. Accordingly, the purpose of this article is to increase the reader's understanding of the use of the qualitative research paradigm in the study of children's experiences with cancer. An overview of four qualitative research designs that pediatric oncology nurse researchers may adopt is presented. Specifically, the qualitative designs of grounded theory, ethnography, phenomenology, and biography or illness narratives are examined. To facilitate discussion, each of the four designs are applied to the study of symptom experiences in children with cancer.

Adaptation, Psychological↗
PubMed2000

Part II: a critical review of qualitative research related to children's experiences with cancer.

Using the qualitative research process to study children's experiences with cancer is being promoted because it is believed that it will afford researchers the opportunity to access children's perspectives of their cancer experiences. A detailed understanding of children's experiences with cancer will result, including a description of their feelings, wants, needs, and concerns. The information gained from qualitative research will help pediatric oncology nurses to better understand what their patients are experiencing. Although adoption of the qualitative paradigm in the study of childhood cancer is in its infancy, qualitative research findings related to children's perspectives of cancer are now slowly emerging. Accordingly, the purpose of this article is to identify some of the meaningful contributions that qualitative research has made in advancing the knowledge base of children's cancer experiences. The first section of this article discusses major themes that have evolved from use of the qualitative research process in the study of children's cancer experiences. This discussion is followed by a critique of the research with suggestions that will aid pediatric nurse researchers in conducting qualitative research when children with cancer are the research participants.

Adaptation, Psychological↗
PubMed1999

Novel human and mouse homologs of Saccharomyces cerevisiae DNA polymerase eta.

The Saccharomyces cerevisiae RAD30 gene encodes a novel eukaryotic DNA polymerase, pol eta that is able to replicate across cis-syn cyclobutane pyrimidine dimers both accurately and efficiently. Very recently, a human homolog of RAD30 was identified, mutations in which result in the sunlight-sensitive, cancer-prone, Xeroderma pigmentosum variant group phenotype. We report here the cloning and localization of a second human homolog of RAD30. Interestingly, RAD30B is localized on chromosome 18q21.1 in a region that is often implicated in the etiology of many human cancers. The mouse homolog (Rad30b) is located on chromosome 18E2. The human RAD30B and mouse Rad30b mRNA transcripts, like many repair proteins, are highly expressed in the testis. In situ hybridization analysis indicates that expression of mouse Rad30b occurs predominantly in postmeiotic round spermatids. Database searches revealed genomic and EST sequences from other eukaryotes such as Aspergillus nidulans, Schizosaccharomyces pombe, Brugia malayi, Caenorhabditis elegans, Trypanosoma cruzi, Arabidopsis thaliana, and Drosophila melanogaster that also encode putative homologs of RAD30, thereby suggesting that Rad30-dependent translesion DNA synthesis is conserved within the eukaryotic kingdom.

Amino Acid Sequence↗
PubMed1999

UmuD'(2)C is an error-prone DNA polymerase, Escherichia coli pol V.

The damage-inducible UmuD' and UmuC proteins are required for most SOS mutagenesis in Escherichia coli. Our recent assay to reconstitute this process in vitro, using a native UmuD'(2)C complex, revealed that the highly purified preparation contained DNA polymerase activity. Here we eliminate the possibility that this activity is caused by a contaminating DNA polymerase and show that it is intrinsic to UmuD'(2)C. E. coli dinB has recently been shown to have DNA polymerase activity (pol IV). We suggest that UmuD'(2)C, the fifth DNA polymerase discovered in E. coli, be designated as E. coli pol V. In the presence of RecA, beta sliding clamp, gamma clamp loading complex, and E. coli single-stranded binding protein (SSB), pol V's polymerase activity is highly "error prone" at both damaged and undamaged DNA template sites, catalyzing efficient bypass of abasic lesions that would otherwise severely inhibit replication by pol III holoenzyme complex (HE). Pol V bypasses a site-directed abasic lesion with an efficiency about 100- to 150-fold higher than pol III HE. In accordance with the "A-rule," dAMP is preferentially incorporated opposite the lesion. A pol V mutant, UmuD'(2)C104 (D101N), has no measurable lesion bypass activity. A kinetic analysis shows that addition of increasing amounts of pol III to a fixed level of pol V inhibits lesion bypass, demonstrating that both enzymes compete for free 3'-OH template-primer ends. We show, however, that despite competition for primer-3'-ends, pol V and pol III HE can nevertheless interact synergistically to stimulate synthesis downstream from a template lesion.

Base Sequence↗
PubMed1999

A phenotype for enigmatic DNA polymerase II: a pivotal role for pol II in replication restart in UV-irradiated Escherichia coli.

DNA synthesis in Escherichia coli is inhibited transiently after UV irradiation. Induced replisome reactivation or "replication restart" occurs shortly thereafter, allowing cells to complete replication of damaged genomes. At the present time, the molecular mechanism underlying replication restart is not understood. DNA polymerase II (pol II), encoded by the dinA (polB) gene, is induced as part of the global SOS response to DNA damage. Here we show that pol II plays a pivotal role in resuming DNA replication in cells exposed to UV irradiation. There is a 50-min delay in replication restart in mutant cells lacking pol II. Although replication restart appears normal in DeltaumuDC strains containing pol II, the restart process is delayed for >90 min in cells lacking both pol II and UmuD'(2)C. Because of the presence of pol II, a transient replication-restart burst is observed in a "quick-stop" temperature-sensitive pol III mutant (dnaE486) at nonpermissive temperature. However, complete recovery of DNA synthesis requires the concerted action of both pol II and pol III. Our data demonstrate that pol II and UmuD'(2)C act in independent pathways of replication restart, thereby providing a phenotype for pol II in the repair of UV-damaged DNA.

Bacterial Proteins↗
PubMed1999

Intermolecular cleavage by UmuD-like enzymes: identification of residues required for cleavage and substrate specificity.

The UmuD-like proteins are best characterized for their role in damage-induced SOS mutagenesis. An essential step in this process is the enzymatic self-processing of the UmuD-like proteins. This reaction is thought to occur either via an intramolecular or intermolecular self-cleavage mechanism. Here, we demonstrate that it can also occur via an heterologous intermolecular cleavage reaction. The Escherichia coli UmuD enzyme demonstrated the broadest substrate specificity, cleaving both E. coli and Salmonella typhimurium UmuD substrates in vivo. In comparison, the wild-type S. typhimurium UmuD (UmuDSt) and MucA enzymes catalyzed intermolecular self-cleavage, but did not facilitate heterologous cleavage. Heterologous cleavage by the UmuDSt enzyme was, however, observed with chimeric UmuD substrates that possess residues 30-55 of UmuDSt. We have further localized the residue predominantly responsible for UmuDSt-catalyzed heterologous cleavage to Ser50 in the substrate molecule. We hypothesize that changes at this residue affect the positioning of the cleavage site of a substrate molecule within the catalytic cleft of the UmuDSt enzyme by affecting the formation of a so-called UmuD "filament-dimer". This hypothesis is further supported by the observation that mutations known to disrupt an E. coli UmuD' filament dimer also block intermolecular UmuDEc cleavage.

Amino Acid Sequence↗
PubMed1999

The bacteriophage P1 HumD protein is a functional homolog of the prokaryotic UmuD'-like proteins and facilitates SOS mutagenesis in Escherichia coli.

The Escherichia coli umuD and umuC genes comprise an operon and encode proteins that are involved in the mutagenic bypass of normally replication-inhibiting DNA lesions. UmuD is, however, unable to function in this process until it undergoes a RecA-mediated cleavage reaction to generate UmuD'. Many homologs of umuDC have now been identified. Most are located on bacterial chromosomes or on broad-host-range R plasmids. One such putative homolog, humD (homolog of umuD) is, however, found on the bacteriophage P1 genome. Interestingly, humD differs from other umuD homologs in that it encodes a protein similar in size to the posttranslationally generated UmuD' protein and not UmuD, nor is it in an operon with a cognate umuC partner. To determine if HumD is, in fact, a bona fide homolog of the prokaryotic UmuD'-like mutagenesis proteins, we have analyzed the ability of HumD to complement UmuD' functions in vivo as well as examined HumD's physical properties in vitro. When expressed from a high-copy-number plasmid, HumD restored cellular mutagenesis and increased UV survival to normally nonmutable recA430 lexA(Def) and UV-sensitive DeltaumuDC recA718 lexA(Def) strains, respectively. Complementing activity was reduced when HumD was expressed from a low-copy-number plasmid, but this observation is explained by immunoanalysis which indicates that HumD is normally poorly expressed in vivo. In vitro analysis revealed that like UmuD', HumD forms a stable dimer in solution and is able to interact with E. coli UmuC and RecA nucleoprotein filaments. We conclude, therefore, that bacteriophage P1 HumD is a functional homolog of the UmuD'-like proteins, and we speculate as to the reasons why P1 might require the activity of such a protein in vivo.

Amino Acid Sequence↗