Errors in the replication of DNA are a major source of spontaneous mutations, and a number of cellular functions are involved in correction of these errors to keep the frequency of spontaneous mutations very low. We report here a novel mechanism which prevents replicational errors by degrading a potent mutagenic substrate for DNA synthesis. This error-avoiding process is catalysed by a protein encoded by the mutT gene of Escherichia coli, mutations of which increase the occurrence of A.T----C.G transversions 100 to 10,000 times the level of the wild type. Spontaneous oxidation of dGTP forms 8-oxo-7,8-dihydro-2'-dGTP (8-oxodGTP), which is inserted opposite dA and dC residues of template DNA with almost equal efficiency, and the MutT protein specifically degrades 8-oxodGTP to the monophosphate. This indicates that elimination from the nucleotide pool of the oxidized form of guanine nucleotide is important for the high fidelity of DNA synthesis.

The ribosomal RNA (rDNA) gene repeats are essential housekeeping genes found in all organisms. A gene amplification system maintains large cluster(s) of tandemly repeated copies in the chromosome, with each species having a specific number of copies. Yeast has many untranscribed rDNA copies (extra copies), and we found that when they are lost, the cells become sensitive to DNA damage induced by mutagens. We show that this sensitivity is dependent on rDNA transcriptional activity, which interferes with cohesion between rDNA loci of sister chromatids. The extra rDNA copies facilitate condensin association and sister-chromatid cohesion, thereby facilitating recombinational repair. These results suggest that high concentrations of heavily transcribed genes are toxic to the cells, and therefore amplified genes, such as rDNA, have evolved.

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Arabidopsis SOG1 (suppressor of gamma response 1) is a plantspecific transcription factor that governs the DNA damage response. Here we report that SOG1 is phosphorylated in response to DNA damage, and that this phosphorylation is mediated by the sensor kinase ataxia telangiectasia mutated (ATM). We show that SOG1 phosphorylation is crucial for the response to DNA damage, including transcriptional induction of downstream genes, transient arrest of cell division and programmed cell death. Although the amino-acid sequences of SOG1 and the mammalian tumour suppressor p53 show no similarity, this study demonstrates that ATM-mediated phosphorylation of a transcription factor has a pivotal role in the DNA damage response in both plants and mammals.

8-0xguanie nucleotide can pair with cytosine and adenine nucleotides at almost equal efficiencies.Once 8-oxodGTP is formed in the cellular nucleotide po-, this mutagenic nucleotide is incorporated into DNA and would cause transversion mutations. The MutT protein ofEscherichia coli possesses enzyme activity to hydrolyze 8-oxodGTP to the corresponding nucleoside monophosphate and thus may be responsible for preventing the occurrence of such mutations. Here we show that the human cell has an enzyme specifically hydrolyzing 8-oxodGTP in a fashion similar to that seen with MutT protein. The human 8-oxodGTPase has been found in cell-free extracts from Jurkat cells and purified >400-fold.Analyses by gel filtration and gel electrophoresis revealed that the molecular mass of the native form of human 8-oxodGTPase is 18 kDa. Mg2+ ion is required for the enzyme action and the optimum pH for the reaction is pH 8.0. The enzyme hydrolyzes 8-oxodGTP to 8-oxodGMP with a K.m value of 12.5 pM. dGTP and dATP are also degraded to dGMP and dAMP, respectively, with Km values 70 times greater than that for 8-oxodGTP. dTTP and dCTP are not hydrolyzed. These properties of the human 8-oxodGTPase are similar to those observed with the E. coli MutT protein, suggesting that the function of protecting the genetic information from the threat of endogenous oxygen radicals is widely distributed in organisms.Mutator mutants that show an increased frequency of spontaneous mutations have led to elucidation of the multiple pathways of spontaneous mutagenesis. Studies on Escherichia coli mutator genes and their products revealed that a major cause of spontaneous mutation is errors of DNA replication and that the cell possesses multistep mechanisms to correct such errors (1). In addition, certain types of spontaneous DNA damage would cause mutations (2) and the cell comes equipped with mechanisms to repair such damage.Among 15 known E. coli mutator genes, 12 are shown to be involved in correction of replicational errors and/or spontaneous DNA damage (1, 3-6). Recently we obtained evidence that the mutT mutator gene is involved in a hitherto unknown mechanism for reducing spontaneous mutation frequency (7).A mutT mutator mutant shows a frequency of A-T -COG transversion 100-10,000 times the level of the wild type (8).The MutT protein specifically prevented misincorporation of dGMP onto poly(dA)/oligo(dT)20 template DNA in vitro (9).This antimutagenic effect of MutT protein appeared to be catalytic and was achieved through its action on dGTP but not on DNA or DNA polymerase (H.M. and M.S., unpublished results). We and others noted that the MutT protein has a weak nucleoside triphosphatase activity with a substrate preference to dGTP (9, 10). Subsequently we found that the nucleotide that is misincorporated opposite the dA residue of the template is not dGMP but rather its oxidized form, 8-oxodGMP. When 8-oxodGTP was added to an in vitro DNA replication system, 8-oxodGMP was incorporated opposite dA and dC residues of the template, with almost e...

SummaryEscherichia coli dinB encodes the specialized DNA polymerase DinB (Pol IV), which is induced as part of the SOS stress-response system and functions in translesion synthesis (TLS) to relieve the replicative Pol III that is stalled at DNA lesions. As the number of DinB molecules, even in unstressed cells, is greater than that required to accomplish TLS, it is thought that dinB plays some additional physiological role. Here, we overexpressed dinB under the tightly regulable arabinose promoter and looked for a distinct phenotype. Upon induction of dinB expression, progression of the replication fork was immediately inhibited at random genomic positions, and the colony-forming ability of the cells was reduced. Overexpression of mutated dinB alleles revealed that the structural requirements for these two inhibitory effects and for TLS were distinct. The extent of in vivo inhibition displayed by a mutant DinB matched the extent of its in vitro impedance, at near-physiological concentration, of a moving Pol III. We suggest that DinB targets Pol III, thereby acting as a brake on replication fork progression. Because the brake operates when cells have excess DinB, as they do under stress conditions, it may serve as a checkpoint that modulates replication to safeguard genome stability.

Background : The inhibition of DNA replication fork progression by DNA lesions can lead to cell death or genome instability. However, little is known about how such DNA lesions affect the concurrent synthesis of leading-and lagging-strand DNA catalysed by the protein machinery used in chromosomal replication. Using a system of semi-bidirectional DNA replication of an oriC plasmid that employs purified replicative enzymes and a replication-terminating protein of Escherichia coli , we examined the dynamics of the replication fork when it encounters a single abasic DNA lesion on the template DNA.

An assay that measures synchronized, processive DNA replication by Escherichia coli DNA polymerase III holoenzyme was used to reveal replacement of pol III by the specialized lesion bypass DNA polymerase IV when the replicative polymerase is stalled. When idled replication is restarted, a rapid burst of pol III-catalyzed synthesis accompanied by ϳ7-kb full-length products is strongly inhibited by the presence of pol IV. The production of slower-forming, shorter length DNA reflects a rapid takeover of DNA synthesis by pol IV. Here we demonstrate that pol IV rapidly (<15 s) obstructs the stable interaction between pol III* and the ␤ clamp (the lifetime of the complex is >5 min), causing the removal of pol III* from template DNA. We propose that the rapid replacement of pol III* on the ␤ clamp with pol IV is mediated by two processes, an interaction between pol IV and the ␤ clamp and a separate interaction between pol IV and pol III*. This newly discovered property of pol IV facilitates a dynamic exchange between the two free polymerases at the primer terminus. Our study suggests a model in which the interaction between pol III* and the ␤ clamp is mediated by pol IV to ensure that DNA replication proceeds with minimal interruption.Successful DNA replication requires a high fidelity DNA polymerase to replicate the entire genome accurately. In Escherichia coli, DNA polymerase III holoenzyme (pol III HE) 2 is a replicative polymerase that catalyzes elongation of DNA chains at a rate of about 1 kb/s with high fidelity. pol III HE is a multisubunit complex that contains two pol III core catalytic subassemblies, each linked to a subunit of the DnaX complex. Polymerase activity resides in the ␣ subunit, the largest subunit of pol III HE. pol III*, a subassembly composed of two pol III cores and one DnaX complex, binds to a dimer of the ␤ subunit (the ␤ clamp) loaded onto the primer/template by the DnaX complex to form a stable structure, pol III HE, which can synthesize DNA processively (over 50 kb per binding event) (1, 2).When a replisome encounters a lesion, pol III is generally believed to stall on DNA at a lesion site on the leading strand, resulting in uncoupling of leading and lagging strand DNA synthesis and arrest of the replication fork (3-5). Two major pathways are thought to play a role in overcoming DNA damage at the replication fork; one is recombinational repair, and the other is the translesion synthesis (TLS). In the TLS pathway, a switch is likely to occur from the stalled replicative polymerase to a specialized polymerase, which replaces the former to bypass the lesion. It is not yet clear, however, how a specialized polymerase gains access to the primer terminus when a stalled replicative polymerase exists at the site or how it acts in coordination with the replicative polymerase (6).In E. coli, three polymerases, pol II, pol IV, and pol V, have been identified as specialized polymerases. pol IV, encoded by the dinB gene, belongs to the Y family and is up-regulated by the SOS response (7). pol IV can r...