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R Woodgate

Publications and source records attributed to R Woodgate.

At least 19 recordsLinked to original sources
PubMed2001

Unique misinsertion specificity of poliota may decrease the mutagenic potential of deaminated cytosines.

DNA polymerase iota (poliota) is a distributive error-prone enzyme that can incorporate nucleotides opposite a variety of DNA lesions. Further elongation is, however, either substantially inhibited or completely abolished. Here, we provide evidence that poliota can facilitate the efficient bypass of uracil and its derivatives as well as oxidized cytosine and guanine residues. The fidelity of translesion replication depends upon the lesion encountered. Correct nucleotides were inserted preferentially opposite 7,8-dihydro-8-oxoguanine (8-oxoG) and 5-hydroxycytosine (5-OHC). However, when bypassing uracil, 5-hydroxyuracil (5-OHU) or 5,6-dihydrouracil (5,6-DHU), poliota inserted T and G with a 4- to 26-fold preference over the Watson-Crick base, A. While the T:U, T:5-OHU and T:5,6-DHU mispairs were extended poorly, the G:U, G:5-OHU and G:5,6-DHU mispairs were extended with equal or greater efficiency than the correctly paired primer termini. Thus, poliota-dependent misinsertion of G opposite uracil and its derivatives may actually provide a mechanism whereby mammalian cells can decrease the mutagenic potential of lesions formed via the deamination of cytosine.

Animals↗
PubMed2001

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): an archaeal DinB-like DNA polymerase with lesion-bypass properties akin to eukaryotic poleta.

Phylogenetic analysis of Y-family DNA polymerases suggests that it can be subdivided into several discrete branches consisting of UmuC/DinB/Rev1/Rad30/Rad30A and Rad30B. The most diverse is the DinB family that is found in all three kingdoms of life. Searches of the complete genome of the crenarchaeon Sulfolobus solfataricus P2 reveal that it possesses a DinB homolog that has been termed DNA polymerase IV (Dpo4). We have overproduced and purified native Dpo4 protein and report here its enzymatic characterization. Dpo4 is thermostable, but can also synthesize DNA at 37 degrees C. Under these conditions, the enzyme exhibits misinsertion fidelities in the range of 8 x 10(-3) to 3 x 10(-4). Dpo4 is distributive but at high enzyme to template ratios can synthesize long stretches of DNA and can substitute for Taq polymerase in PCR. On damaged DNA templates, Dpo4 can facilitate translesion replication of an abasic site, a cis-syn thymine-thymine dimer, as well as acetyl aminofluorene adducted- and cisplatinated-guanine residues. Thus, although phylogenetically related to DinB polymerases, our studies suggest that the archaeal Dpo4 enzyme exhibits lesion-bypass properties that are, in fact, more akin to those of eukaryotic poleta.

Archaeal Proteins↗
PubMed2001

Crystal structure of a Y-family DNA polymerase in action: a mechanism for error-prone and lesion-bypass replication.

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) is a DinB homolog that belongs to the recently described Y-family of DNA polymerases, which are best characterized by their low-fidelity synthesis on undamaged DNA templates and propensity to traverse normally replication-blocking lesions. Crystal structures of Dpo4 in ternary complexes with DNA and an incoming nucleotide, either correct or incorrect, have been solved at 1.7 A and 2.1 A resolution, respectively. Despite a conserved active site and a hand-like configuration similar to all known polymerases, Dpo4 makes limited and nonspecific contacts with the replicating base pair, thus relaxing base selection. Dpo4 is also captured in the crystal translocating two template bases to the active site at once, suggesting a possible mechanism for bypassing thymine dimers.

Amino Acid Sequence↗
PubMed2001

Roles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coli.

DNA polymerase V, composed of a heterotrimer of the DNA damage-inducible UmuC and UmuD(2)(') proteins, working in conjunction with RecA, single-stranded DNA (ssDNA)-binding protein (SSB), beta sliding clamp, and gamma clamp loading complex, are responsible for most SOS lesion-targeted mutations in Escherichia coli, by catalyzing translesion synthesis (TLS). DNA polymerase II, the product of the damage-inducible polB (dinA ) gene plays a pivotal role in replication-restart, a process that bypasses DNA damage in an error-free manner. Replication-restart takes place almost immediately after the DNA is damaged (approximately 2 min post-UV irradiation), whereas TLS occurs after pol V is induced approximately 50 min later. We discuss recent data for pol V-catalyzed TLS and pol II-catalyzed replication-restart. Specific roles during TLS for pol V and each of its accessory factors have been recently determined. Although the precise molecular mechanism of pol II-dependent replication-restart remains to be elucidated, it has recently been shown to operate in conjunction with RecFOR and PriA proteins.

Catalysis↗
PubMed2001

Human DNA polymerase iota promiscuous mismatch extension.

Human DNA polymerase iota is a low-fidelity template copier that preferentially catalyzes the incorporation of the wobble base G, rather than the Watson-Crick base A, opposite template T (Tissier, A., McDonald, J. P., Frank, E. G., and Woodgate, R. (2000) Genes Dev. 14, 1642-1650; Johnson, R. E., Washington, M. T., Haracska, L., Prakash, S., and Prakash, L. (2000) Nature 406, 1015-1019; Zhang, Y., Yuan, F., Wu, X., and Wang, Z. (2000) Mol. Cell. Biol. 20, 7099-7108). Here, we report on its ability to extend all 12 possible mispairs and 4 correct pairs in different sequence contexts. Extension from both matched and mismatched primer termini is generally most efficient and accurate when A is the next template base. In contrast, extension occurs less efficiently and accurately when T is the target template base. A striking exception occurs during extension of a G:T mispair, where the enzyme switches specificity, "preferring" to make a correct A:T base pair immediately downstream from an originally favored G:T mispair. Polymerase iota generates a variety of single and tandem mispairs with high frequency, implying that it may act as a strong mutator when copying undamaged DNA templates in vivo. Even so, its limited ability to catalyze extension from a relatively stable primer/template containing a "buried" mismatch suggests that polymerase iota-catalyzed errors are confined to short template regions.

Base Pair Mismatch↗
PubMed2001

Altered nucleotide misinsertion fidelity associated with poliota-dependent replication at the end of a DNA template.

A hallmark of human DNA polymerase iota (poliota) is the asymmetric fidelity of replication at template A and T when the enzyme extends primers annealed to a single-stranded template. Here, we report on the efficiency and accuracy of poliota-dependent replication at a nick, a gap, the very end of a template and from a mispaired primer. Poliota cannot initiate synthesis on a nicked DNA substrate, but fills short gaps efficiently. Surprisingly, poliota's ability to blunt-end a 1 bp recessed terminus is dependent upon the template nucleotide encountered and is highly erroneous. At template G, both C and T are inserted with roughly equal efficiency, whilst at template C, C and A are misinserted 8- and 3-fold more often than the correct base, G. Using substrates containing mispaired primer termini, we show that poliota can extend all 12 mispairs, but with differing efficiencies. Poliota can also extend a tandem mispair, especially when it is located within a short gap. The enzymatic properties of poliota appear consistent with that of a somatic hypermutase and suggest that poliota may be one of the low-fidelity DNA polymerases hypothesized to participate in the hypermutation of immunoglobulin variable genes in vivo.

Animals↗
PubMed2001

5'-Deoxyribose phosphate lyase activity of human DNA polymerase iota in vitro.

DNA polymerase iota (pol iota) is one of several recently discovered DNA polymerases in mammalian cells whose function is unknown. We report here that human pol iota has an intrinsic 5'-deoxyribose phosphate (dRP) lyase activity. In reactions reconstituted with uracil-DNA glycosylase (UDG), apurinic/apyrimidinic (AP) endonuclease and DNA ligase I, pol iota can use its dRP lyase and polymerase activities to repair G*U and A*U pairs in DNA. These data and three distinct catalytic properties of pol iota implicate it in specialized forms of base excision repair (BER).

Amino Acid Sequence↗
PubMed2001

Evolution of the two-step model for UV-mutagenesis.

It is quite remarkable how our understanding of translesion DNA synthesis (TLS) has changed so dramatically in the past 2 years. Until very recently, little was known about the molecular mechanisms of TLS in higher eukaryotes and what we did know, was largely based upon Escherichia coli and Saccharomyces cerevisiae model systems. The paradigm, proposed by Bryn Bridges and I [Mutat. Res. 150 (1985) 133] in 1985, was that error-prone TLS occurred in two steps; namely a misinsertion event opposite a lesion, followed by extension of the mispair so as to facilitate complete bypass of the lesion. The initial concept was that at least for E. coli, the misinsertion event was performed by the cell's main replicase, DNA polymerase III holoenzyme, and that elongation was achieved through the actions of specialized polymerase accessory proteins, such as UmuD and UmuC. Some 15 years later, we now know that this view is likely to be incorrect in that both misinsertion and bypass are performed by the Umu proteins (now called pol V). As pol V is normally a distributive enzyme, pol III may only be required to "fix" the misincorporation as a mutation by completing chromosome duplication. However, while the role of the E. coli proteins involved in TLS have changed, the initial concept of misincorporation followed by extension/bypass remains valid. Indeed, recent evidence suggests that it can equally be applied to TLS in eukaryotic cells where there are many more DNA polymerases to choose from. The aim of this review is, therefore, to provide a historical perspective to the "two-step" model for UV-mutagenesis, how it has recently evolved, and in particular, to highlight the seminal contributions made to it by Bryn Bridges.

DNA Polymerase III↗
PubMed2001

DNA polymerase iota and related rad30-like enzymes.

Until recently, the molecular mechanisms of translesion DNA synthesis (TLS), a process whereby a damaged base is used as a template for continued replication, was poorly understood. This area of scientific research has, however, been revolutionized by the finding that proteins long implicated in TLS are, in fact, DNA polymerases. Members of this so-called UmuC/DinB/Rev1/Rad30 superfamily of polymerases have been identified in prokaryotes, eukaryotes and archaea. Biochemical studies with the highly purified polymerases reveal that some, but not all, can traverse blocking lesions in template DNA. All of them share a common feature, however, in that they exhibit low fidelity when replicating undamaged DNA. Of particular interest to us is the Rad30 subfamily of polymerases found exclusively in eukaryotes. Humans possess two Rad30 paralogs, Rad30A and Rad30B. The RAD30A gene encodes DNA polymerase eta and defects in the protein lead to the xeroderma pigmentosum variant (XP-V) phenotype in humans. Very recently RAD30B has also been shown to encode a novel DNA polymerase, designated as Pol iota. Based upon in vitro studies, it appears that Pol iota has the lowest fidelity of any eukaryotic polymerase studied to date and we speculate as to the possible cellular functions of such a remarkably error-prone DNA polymerase.

Bacterial Proteins↗
PubMed2001

A model for SOS-lesion-targeted mutations in Escherichia coli.

The UmuD'2C protein complex (Escherichia coli pol V) is a low-fidelity DNA polymerase (pol) that copies damaged DNA in the presence of RecA, single-stranded-DNA binding protein (SSB) and the beta,gamma-processivity complex of E. coli pol III (ref. 4). Here we propose a model to explain SOS-lesion-targeted mutagenesis, assigning specific biochemical functions for each protein during translesion synthesis. (SOS lesion-targeted mutagenesis occurs when pol V is induced as part of the SOS response to DNA damage and incorrectly incorporates nucleotides opposite template lesions.) Pol V plus SSB catalyses RecA filament disassembly in the 3' to 5' direction on the template, ahead of the polymerase, in a reaction that does not involve ATP hydrolysis. Concurrent ATP-hydrolysis-driven filament disassembly in the 5' to 3' direction results in a bidirectional stripping of RecA from the template strand. The bidirectional collapse of the RecA filament restricts DNA synthesis by pol V to template sites that are proximal to the lesion, thereby minimizing the occurrence of untargeted mutations at undamaged template sites.

Adenosine Triphosphate↗
PubMed2001

Biochemical characterization of human DNA polymerase iota provides clues to its biological function.

The human RAD30B gene has recently been shown to encode a novel DNA polymerase, DNA polymerase iota (poliota). The role of poliota within the cell is presently unknown, and the only clues to its cellular function come from its biochemical characterization in vitro. The aim of this short review is, therefore, to summarize the known enzymic activities of poliota and to speculate as to how these biochemical properties might relate to its in vivo function.

Base Pair Mismatch↗
PubMed2001

Adopting the qualitative paradigm to understanding children's perspectives of illness: barrier or facilitator?

Reliance on the traditional quantitative paradigm has been the norm for exploring the phenomenon of children's illness experiences. Although it provides valuable information on the psychosocial sequelae of children in response to illness, an account of children's perspectives of the illness experience is missing. Accordingly, researchers since the early 1980s have been advocating the need to adopt the qualitative paradigm in the study of children's illness experiences. Nonetheless, even though there is a call for more qualitative research, the majority of research being conducted today still supports the quantitative paradigm. This report examines why researchers may be reluctant to adopt the qualitative paradigm. Specifically, the author proposes that this reluctance may stem from researchers' perceiving children as being less competent and social than adults, which would explain the need to research children solely from an objective, impersonal stance. Primary philosophical principles of the qualitative paradigm may be seen as barriers to accessing children's thoughts. The author suggests, however, that when children are viewed from an alternative perspective, the major principles of the qualitative paradigm may actually serve as facilitators to apprehending their thoughts and feelings. The following key principles of the qualitative research paradigm are examined: (a) realities are constructed by human beings who are viewed as active agents making sense out of the realities they encounter; (b) realities are multiple and must be perceived holistically and from various vantage points; and (c) realities are shared and mutually shaped by the researcher and research participants. Key strategies that promote successful use of the qualitative paradigm in the study of children's illness experiences are also discussed.

Attitude of Health Personnel↗
PubMed2001

Molecular analysis of antibiotic resistance gene clusters in vibrio cholerae O139 and O1 SXT constins.

Many recent Asian clinical Vibrio cholerae E1 Tor O1 and O139 isolates are resistant to the antibiotics sulfamethoxazole (Su), trimethoprim (Tm), chloramphenicol (Cm), and streptomycin (Sm). The corresponding resistance genes are located on large conjugative elements (SXT constins) that are integrated into prfC on the V. cholerae chromosome. We determined the DNA sequences of the antibiotic resistance genes in the SXT constin in MO10, an O139 isolate. In SXT(MO10), these genes are clustered within a composite transposon-like structure found near the element's 5' end. The genes conferring resistance to Cm (floR), Su (sulII), and Sm (strA and strB) correspond to previously described genes, whereas the gene conferring resistance to Tm, designated dfr18, is novel. In some other O139 isolates the antibiotic resistance gene cluster was found to be deleted from the SXT-related constin. The El Tor O1 SXT constin, SXT(ET), does not contain the same resistance genes as SXT(MO10). In this constin, the Tm resistance determinant was located nearly 70 kbp away from the other resistance genes and found in a novel type of integron that constitutes a fourth class of resistance integrons. These studies indicate that there is considerable flux in the antibiotic resistance genes found in the SXT family of constins and point to a model for the evolution of these related mobile elements.

Bacterial Proteins↗
PubMed2001

Formation of chromosomal tandem arrays of the SXT element and R391, two conjugative chromosomally integrating elements that share an attachment site.

The SXT element, a conjugative, self-transmissible, integrating element (a constin) originally derived from a Vibrio cholerae O139 isolate from India, and IncJ element R391, originally derived from a South African Providencia rettgeri isolate, were found to be genetically and functionally related. Both of these constins integrate site specifically into the Escherichia coli chromosome at an identical attachment site within the 5' end of prfC. They encode nearly identical integrases, which are required for chromosomal integration, excision, and extrachromosomal circularization of these elements, and they have similar tra genes. Therefore, these closely related constins have virtually identical mechanisms for chromosomal integration and dissemination. The presence of either element in a recipient cell did not significantly reduce its ability to acquire the other element, indicating that R391 and SXT do not encode surface exclusion determinants. In cells harboring both elements, SXT and R391 were integrated in tandem fashion on the chromosome, and homologous recombination appeared to play little or no role in the formation of these arrays. Interference between R391 and SXT was detected by measuring the frequency of loss of an unselected resident element upon introduction of a second selected element. In these assays, R391 was found to have a stronger effect on SXT stability than vice versa. The level of expression and/or activity of the donor and recipient integrases may play a role in the interference between these two related constins.

Chromosomes, Bacterial↗
PubMed2000

Identification of mucAB-like homologs on two IncT plasmids, R394 and Rts-1.

Recent phylogenetic analysis of the superfamily of lesion-replicating DNA polymerases suggest that they can be broadly divided into four sub-groups comprised of UmuC-like, DinB-like, Rev1-like and Rad30-like proteins. The UmuC-like sub-family is best characterized at the genetic level and sequence analysis of eleven umu orthologs, residing on bacterial chromosomes or on self-transmissible R-plasmids allows further subdivision into five sub-groups (UmuDC, MucAB, ImpAB, RumAB and RulAB) based on amino acid sequence conservation. Some of these orthologs are apparently inactive in situ, but may promote increased mutagenesis and survival when subcloned and expressed from high-copy number plasmids. We were, therefore, interested in devising an assay that would identify umuC-like genes in situ in the absence of a functional assay. To this end, degenerate primers directed towards conserved amino acid regions within the UmuC-like sub-family of DNA polymerases were designed and used to identify mucAB-like operons on the IncT plasmids, R394 and Rts-1.Interestingly, DNA sequence analysis of an approximately 7kb region of R394 identified two LexA-regulated genes immediately downstream of mucAB((R394)) that are similar to the chromosomally-encoded Escherichia coli tus gene and the IncI plasmid-encoded impC gene, respectively. Analysis of the R394 and Rts-1 mucB genes revealed that both contain insertions which result in the expression of a truncated inactive MucB protein. While R394 was unable to restore mutagenesis functions to a DeltaumuDC E. coli strain, Rts-1 surprisingly promoted significant levels of MMS-induced SOS mutagenesis, raising the possibility that Rts-1 encodes another, yet unidentified, umu-like homolog.

Amino Acid Sequence↗
PubMed2000

Subunit-specific degradation of the UmuD/D' heterodimer by the ClpXP protease: the role of trans recognition in UmuD' stability.

The Escherichia coli UmuD' protein is a subunit of the recently described error-prone DNA polymerase, pol V. UmuD' is initially synthesized as an unstable and mutagenically inactive pro-protein, UmuD. Upon processing, UmuD' assumes a relatively stable conformation and becomes mutagenically active. While UmuD and UmuD' by themselves exist in vivo as homodimers, when together they preferentially interact to form heterodimers. Quite strikingly, it is in this context that UmuD' becomes susceptible to ClpXP-mediated proteolysis. Here we report a novel targeting mechanism designed for degrading the mutagenically active UmuD' subunit of the UmuD/D' heterodimer complex, while leaving the UmuD protein intact. Surprisingly, a signal that is essential and sufficient for targeting UmuD' for degradation was found to reside on UmuD not UmuD'. UmuD was also shown to be capable of channeling an excess of UmuD' to ClpXP for degradation, thereby providing a mechanism whereby cells can limit error-prone DNA replication.

Adenosine Triphosphatases↗