Mathematical model of L5178Y mouse lymphoma forward mutation assay

A mathematical model of the biological protocol for the Mouse Lymphoma L5178Y Forward Mutation Bioassay is presented. The model relates the mutant progenitor frequency (MPF), the number of cells per million surviving cells with DNA damage after exposure to the chemical, to the mutant frequency (MF), the number of TFT-resistant cells per million survivors. For a given expression time, the deterministic relationship is linear and the proportionality constant depends on the relative suspension growth factor (rg) and relative cloning efficiencies (rc) of mutants to those of wild type cells: MF = (rg X rc) X MPF. Experimental noise leads to variations in the values of rg and rc and lack of reproducibility in the system. If mutant progenitors and their progeny grow as well as wild-type cells and if all of the parental mutant progenitors express the mutant phenotype, then rg = 1/2 and rc = 1. Biological mechanisms, such as differential growth characteristics of mutant and wild-type cells or DNA repair, can make the mutant frequency an inaccurate estimate of the MPF. For the assay to be useful as a screen for the mutagenic activity of chemicals, rg X rc has to be reasonably constant from chemical to chemical.     

The 3'-->5' exonucleases of DNA polymerases delta and epsilon and the 5'-->3' exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae

Replication fidelity is controlled by DNA polymerase proofreading and postreplication mismatch repair. We have genetically characterized the roles of the 5'-->3' Exo1 and the 3'-->5' DNA polymerase exonucleases in mismatch repair in the yeast Saccharomyces cerevisiae by using various genetic backgrounds and highly sensitive mutation detection systems that are based on long and short homonucleotide runs. Genetic interactions were examined among DNA polymerase epsilon (pol2-4) and delta (pol3-01) mutants defective in 3'-->5' proofreading exonuclease, mutants defective in the 5'-->3' exonuclease Exo1, and mismatch repair mutants (msh2, msh3, or msh6). These three exonucleases play an important role in mutation avoidance. Surprisingly, the mutation rate in an exo1 pol3-01 mutant was comparable to that in an msh2 pol3-01 mutant, suggesting that they participate directly in postreplication mismatch repair as well as in other DNA metabolic processes.     

Structure of the MutL C-terminal domain: a model of intact MutL and its roles in mismatch repair

MutL assists the mismatch recognition protein MutS to initiate and coordinate mismatch repair in species ranging from bacteria to humans. The MutL N-terminal ATPase domain is highly conserved, but the C-terminal region shares little sequence similarity among MutL homologs. We report here the crystal structure of the Escherichia coli MutL C-terminal dimerization domain and the likelihood of its conservation among MutL homologs. A 100-residue proline-rich linker between the ATPase and dimerization domains, which generates a large central cavity in MutL dimers, tolerates sequence substitutions and deletions of one-third of its length with no functional consequences in vivo or in vitro. Along the surface of the central cavity, residues essential for DNA binding are located in both the N- and C-terminal domains. Each domain of MutL interacts with UvrD helicase and is required for activating the helicase activity. The DNA-binding capacity of MutL is correlated with the level of UvrD activation. A model of how MutL utilizes its ATPase and DNA-binding activities to mediate mismatch-dependent activation of MutH endonuclease and UvrD helicase is proposed.     

The extreme mutator effect of Escherichia coli mutD5 results from saturation of mismatch repair by excessive DNA replication errors.

Escherichia coli mutator mutD5 is the most potent mutator known. The mutD5 mutation resides in the dnaQ gene encoding the proofreading exonuclease of DNA polymerase III holoenzyme. It has recently been shown that the extreme mutability of this strain results, in addition to a proofreading defect, from a defect in mutH, L, S-encoded postreplicational DNA mismatch repair. The following measurements of the mismatch-repair capacity of mutD5 cells demonstrate that this mismatch-repair defect is not structural, but transient. mutD5 cells in early log phase are as deficient in mismatch repair as mutL cells, but they become as proficient as wild-type cells in late log phase. Second, arrest of chromosomal replication in a mutD5-dnaA(Ts) strain at a nonpermissive temperature restores mismatch repair, even from the early log phase of growth. Third, transformation of mutD5 strains with multicopy plasmids expressing the mutH or mutL gene restores mismatch repair, even in rapidly growing cells. These observations suggest that the mismatch-repair deficiency of mutD strains results from a saturation of the mutHLS-mismatch-repair system by an excess of primary DNA replication errors due to the proofreading defect.     

DNA合成的忠实性机制

DNA的忠实性合成对于基因组稳定和物种延续至关重要,否则可能会产生严重的后果。DNA合成具有极高的忠实性,这主要基于3个步骤:(1)基于氢键、碱基对构象或其他因素的核苷酸选择;(2)基于3′→5′外切酶活性的校对,方式有顺式校对和反式校对,可以去除错误掺入的核苷酸;(3)基于错配修复、切除修复、同源重组修复和跨损伤DNA合成的修复过程,可以纠正逃过校对的错误核苷酸。由于DNA聚合酶不仅可以作为抗病毒或抗癌药物的靶标,而且其忠实性还与抗药性或药物副作用有关,所以深入研究DNA合成的忠实性具有非常重要的意义。文章主要论述了DNA合成的忠实性机制,并对DNA聚合酶的应用前景做了展望。     

Cytostatic, cytotoxic and mutagenic effects of voacristine, an indole alkaloid in wild-type and repair-deficient yeasts

Voacristine, an indole alkaloid isolated from the leaves of Ervatamia coronaria (Stapf.) (Apocynaceae) has dose-dependent cytostatic and cytotoxic effects on cultures of Saccharomyces cerevisiae. These inhibitory effects take place only in growing cells. Among the different repair-deficient mutants examined, a mutant defective in excision-resynthesis repair pathway (rad3-e5) was found to be the most sensitive to such a toxic effect. The mutant rad52-1 blocked in the DNA strand break repair pathway showed an intermediary sensitivity to the lethal effect induced by this indole alkaloid, whereas the mutant defective in the mutagenic repair pathway (rad6-1) demonstrated practically the same sensitivity as the wild-type strain. The nuclear reversion mutation for the locus lysl-1 was induced by voacristine, whereas the mitochondrial "petite" mutation was not induced by this alkaloid. These results indicate that the lesions induced by voacristine in vivo are likely to be of the adducts type; such damage is repairable in the wild-type; the DNA strand break repair pathway plays a minor role in the repair of voacristine-induced lesions.     

Inhibition of the Jun kinase pathway blocks DNA repair, enhances p53-mediated apoptosis and promotes gene amplification.

We have previously shown, by expression of a nonphosphorylatable dominant inhibitor mutant of c-Jun [cJun(S63A,S73A)], that activation of the NH2-terminal Jun kinase/stress-activated protein kinase by genotoxic damage is required for DNA repair. Here, we examine the consequences of inhibition of DNA repair on p53-induced apoptosis in T98G cells, which are devoid of endogenous wild-type p53. Relative to parental or wild-type c-Jun-expressing control cells, mutant Jun-expressing T98G clones show similar growth rates and plating efficiencies. However, these cells are unable to repair DNA (PCR-stop assays) and exhibit up to an 80-fold increased methotrexate-induced colony formation due to amplification of the dihydrofolate reductase gene. Moreover, the mutant c-Jun clones exhibit increased apoptosis and elevated bax:bcl2 ratios on expression of wild-type p53. These results indicate that inhibition of DNA repair leads to accumulation of DNA damage in tumor cells with unstable genomes and this, in turn, enhances p53mediated apoptosis.     

Genetic factors affecting the impact of DNA polymerase delta proofreading activity on mutation avoidance in yeast

Base selectivity, proofreading, and postreplication mismatch repair are important for replication fidelity. Because proofreading plays an important role in error correction, we have investigated factors that influence its impact in the yeast Saccharomyces cerevisiae. We have utilized a sensitive mutation detection system based on homonucleotide runs of 4 to 14 bases to examine the impact of DNA polymerase delta proofreading on mutation avoidance. The contribution of DNA polymerase delta proofreading on error avoidance was found to be similar to that of DNA polymerase epsilon proofreading in short homonucleotide runs (A4 and A5) but much greater than the contribution of DNA polymerase epsilon proofreading in longer runs. We have identified an intraprotein interaction affecting mutation prevention that results from mutations in the replication and the proofreading regions, resulting in an antimutator phenotype relative to a proofreading defect. Finally, a diploid strain with a defect in DNA polymerase delta proofreading exhibits a higher mutation rate than a haploid strain. We suggest that in the diploid population of proofreading defective cells there exists a transiently hypermutable fraction that would be inviable if cells were haploids.     

Effect of proofreading and dam-instructed mismatch repair systems on N4-hydroxycytidine-induced mutagenesis

Summary The role of the proofreading (35 exonuclease) function of T4 DNA polymerase and the mismatch repair system ofE. coli on N⁴-hydroxycytidine (oh⁴Cyd)¹ induced mutagenesis was investigated. oh⁴Cyd-induced mutation is strongly suppressed when the proofreading activity increases as a result of the presence oftsCB87-antimutator polymerase or elevated temperature (43° C vs 30° C). Mutagenic activity of oh⁴Cyd, however, is little, if at all, affected by the presence of thetsLB56 mutator allele of T4 DNA polymerase with suppressed proofreading activity. This leads to the conclusion that oh⁴C nucleotides are not frequently removed by proofreading activity of wild-type T4 DNA polymerase. The number of mutations induced by oh⁴Cyd increases 3- to 5-fold due to damage of the genesmutS,mutL,uvrE, but notmutR.Dam ^- cells are more sensitive to, and hypermutable by, oh⁴Cyd in comparison withdam ⁺ cells. This is compatible with the notion that oh⁴C residues are recognised and excised by mismatch repair enzymes. The results indicate thath neither the proofreading function of T4 DNA polymerase, nor the mismatch repair enzymes, are responsible for the high specificity of oh⁴Cyd which causes ATGC transition.     

Fragile DNA repair mechanism reduces ageing in multicellular model

DNA damages, as well as mutations, increase with age. It is believed that these result from increased genotoxic stress and decreased capacity for DNA repair. The two causes are not independent, DNA damage can, for example, through mutations, compromise the capacity for DNA repair, which in turn increases the amount of unrepaired DNA damage. Despite this vicious circle, we ask, can cells maintain a high DNA repair capacity for some time or is repair capacity bound to continuously decline with age? We here present a simple mathematical model for ageing in multicellular systems where cells subjected to DNA damage can undergo full repair, go apoptotic, or accumulate mutations thus reducing DNA repair capacity. Our model predicts that at the tissue level repair rate does not continuously decline with age, but instead has a characteristic extended period of high and non-declining DNA repair capacity, followed by a rapid decline. Furthermore, the time of high functionality increases, and consequently slows down the ageing process, if the DNA repair mechanism itself is vulnerable to DNA damages. Although counterintuitive at first glance, a fragile repair mechanism allows for a faster removal of compromised cells, thus freeing the space for healthy peers. This finding might be a first step toward understanding why a mutation in single DNA repair protein (e.g. Wrn or Blm) is not buffered by other repair proteins and therefore, leads to severe ageing disorders.     

A biological-based model that links genomic instability, bystander effects, and adaptive response

This paper links genomic instability, bystander effects, and adaptive response in mammalian cell communities via a novel biological-based, dose-response model called NEOTRANS3. The model is an extension of the NEOTRANS2 model that addressed stochastic effects (genomic instability, mutations, and neoplastic transformation) associated with brief exposure to low radiation doses. With both models, ionizing radiation produces DNA damage in cells that can be associated with varying degrees of genomic instability. Cells with persistent problematic instability (PPI) are mutants that arise via misrepair of DNA damage. Progeny of PPI cells also have PPI and can undergo spontaneous neoplastic transformation. Unlike NEOTRANS2, with NEOTRANS3 newly induced mutant PPI cells and their neoplastically transformed progeny can be suppressed via our previously introduced protective apoptosis-mediated (PAM) process, which can be activated by low linear energy transfer (LET) radiation. However, with NEOTRANS3 (which like NEOTRANS2 involves cross-talk between nongenomically compromised [e.g., nontransformed, nonmutants] and genomically compromised [e.g., mutants, transformants, etc.] cells), it is assumed that PAM is only activated over a relatively narrow, dose-rate-dependent interval (D(PAM),D(off)); where D(PAM) is a small stochastic activation threshold, and D(off) is the stochastic dose above which PAM does not occur. PAM cooperates with activated normal DNA repair and with activated normal apoptosis in guarding against genomic instability. Normal repair involves both error-free repair and misrepair components. Normal apoptosis and the error-free component of normal repair protect mammals by preventing the occurrence of mutant cells. PAM selectively removes mutant cells arising via the misrepair component of normal repair, selectively removes existing neoplastically transformed cells, and probably selectively removes other genomically compromised cells when it is activated. PAM likely involves multiple pathways to apoptosis, with the selected pathway depending on the type of cell to be removed, its cellular environment, and on the nature of the genomic damage.     

Partial reconstitution of human DNA mismatch repair in vitro: characterization of the role of human replication protein A

DNA mismatch repair (MMR) is a critical genome-stabilization system. However, the molecular mechanism of MMR in human cells remains obscure because many of the components have not yet been identified. Using a functional in vitro reconstitution system, this study identified three HeLa cell fractions essential for in vitro MMR. These fractions divide human MMR into two distinct stages: mismatch-provoked excision and repair synthesis. In vitro dissection of the MMR reaction and crucial intermediates elucidated biochemical functions of individual fractions in human MMR and identified hitherto unknown functions of human replication protein A (hRPA) in MMR. Thus, one fraction carries out nick-directed and mismatch-dependent excision; the second carries out DNA repair synthesis and DNA ligation; and the third provides hRPA, which plays multiple roles in human MMR by protecting the template DNA strand from degradation, enhancing repair excision, and facilitating repair synthesis. It is anticipated that further analysis of these fractions will identify additional MMR components and enable the complete reconstitution of the human MMR pathway with purified proteins.     

Camptothecin enhances the frequency of oligonucleotide-directed gene repair in mammalian cells by inducing DNA damage and activating homologous recombination

Camptothecin (CPT) is an anticancer drug that promotes DNA breakage at replication forks and the formation of lesions that activate the processes of homologous recombination (HR) and nonhomologous end joining. We have taken advantage of the CPT-induced damage response by coupling it to gene repair directed by synthetic oligonucleotides, a process in which a mutant base pair is converted into a wild-type one. Here, we show that pretreating DLD-1 cells with CPT leads to a significant stimulation in the frequency of correction of an integrated mutant enhanced green fluorescent protein gene. The stimulation is dose-dependent and coincident with the formation of double-strand DNA breaks. Caffeine, but not vanillin, blocks the enhancement of gene repair suggesting that, in this system, HR is the pathway most responsible for elevating the frequency of correction. The involvement of HR is further proven by studies in which wortmannin was seen to inhibit gene repair at high concentrations but not at lower levels that are known to inhibit DNA-PK activity. Taken together, our results suggest that DNA damage induced by CPT activates a cellular response that stimulates gene repair in mammalian cells.     

Identification of a mutant DNA polymerase delta in Saccharomyces cerevisiae with an antimutator phenotype for frameshift mutations

We propose that a beta-turn-beta structure, which plays a critical role in exonucleolytic proofreading in the bacteriophage T4 DNA polymerase, is also present in the Saccharomyces cerevisiae DNA pol delta. Site-directed mutagenesis was used to test this proposal by introducing a mutation into the yeast POL3 gene in the region that encodes the putative beta-turn-beta structure. The mutant DNA pol delta has a serine substitution in place of glycine at position 447. DNA replication fidelity of the G447S-DNA pol delta was determined in vivo by using reversion and forward assays. An antimutator phenotype for frameshift mutations in short homopolymeric tracts was observed for the G447S-DNA pol delta in the absence of postreplication mismatch repair, which was produced by inactivation of the MSH2 gene. Because the G447S substitution reduced frameshift but not base substitution mutagenesis, some aspect of DNA polymerase proofreading appears to contribute to production of frameshifts. Possible roles of DNA polymerase proofreading in frameshift mutagenesis are discussed.     

Mismatch repair-independent increase in spontaneous mutagenesis in yeast lacking non-essential subunits of DNA polymerase ε

Yeast DNA polymerase ε (Pol ε) is a highly accurate and processive enzyme that participates in nuclear DNA replication of the leading strand template. In addition to a large subunit (Pol2) harboring the polymerase and proofreading exonuclease active sites, Pol ε also has one essential subunit (Dpb2) and two smaller, non-essential subunits (Dpb3 and Dpb4) whose functions are not fully understood. To probe the functions of Dpb3 and Dpb4, here we investigate the consequences of their absence on the biochemical properties of Pol ε in vitro and on genome stability in vivo. The fidelity of DNA synthesis in vitro by purified Pol2/Dpb2, i.e. lacking Dpb3 and Dpb4, is comparable to the four-subunit Pol ε holoenzyme. Nonetheless, deletion of DPB3 and DPB4 elevates spontaneous frameshift and base substitution rates in vivo, to the same extent as the loss of Pol ε proofreading activity in a pol2-4 strain. In contrast to pol2-4, however, the dpb3Δdpb4Δ does not lead to a synergistic increase of mutation rates with defects in DNA mismatch repair. The increased mutation rate in dpb3Δdpb4Δ strains is partly dependent on REV3, as well as the proofreading capacity of Pol δ. Finally, biochemical studies demonstrate that the absence of Dpb3 and Dpb4 destabilizes the interaction between Pol ε and the template DNA during processive DNA synthesis and during processive 3' to 5'exonucleolytic degradation of DNA. Collectively, these data suggest a model wherein Dpb3 and Dpb4 do not directly influence replication fidelity per se, but rather contribute to normal replication fork progression. In their absence, a defective replisome may more frequently leave gaps on the leading strand that are eventually filled by Pol ζ or Pol δ, in a post-replication process that generates errors not corrected by the DNA mismatch repair system.     

Rat DNA polymerase beta gene can join in excision repair of Escherichia coli.

Though DNA polymerase I (poll) of Escherichia (E.) coli is understood to play a role in repair synthesis of excision repair, it is still obscure whether DNA polymerase beta (pol beta) plays a similar role in eukaryotic cells. To estimate the role of pol beta in excision repair processes, we inserted the rat pol beta gene into several mutant E. coli defective in a diverse set of enzymatic activities of poll. UV resistance was seen only when the 5'----3' exonuclease (exo) activity of poll molecules remained. Therefore it is suggested that 5'----3' exo activity as well as pol beta activity are essential for repair synthesis of excision repair in eukaryotic cells.     

Human Pol ɛ-dependent replication errors and the influence of mismatch repair on their correction

Mutations in human DNA polymerase (Pol) ?, one of three eukaryotic Pols required for DNA replication, have recently been found associated with an ultramutator phenotype in tumors from somatic colorectal and endometrial cancers and in a familial colorectal cancer. Possibly, Pol ? mutations reduce the accuracy of DNA synthesis, thereby increasing the mutational burden and contributing to tumor development. To test this possibility in vivo, we characterized an active site mutant allele of human Pol ? that exhibits a strong mutator phenotype in vitro when the proofreading exonuclease activity of the enzyme is inactive. This mutant has a strong bias toward mispairs opposite template pyrimidine bases, particularly T•dTTP mispairs. Expression of mutant Pol ? in human cells lacking functional mismatch repair caused an increase in mutation rate primarily due to T•dTTP mispairs. Functional mismatch repair eliminated the increased mutagenesis. The results indicate that the mutant Pol ? causes replication errors in vivo, and is at least partially dominant over the endogenous, wild type Pol ?. Since tumors from familial and somatic colorectal patients arise with Pol ? mutations in a single allele, are microsatellite stable and have a large increase in base pair substitutions, our data are consistent with a Pol ? mutation requiring additional factors to promote tumor development.     

Growth-phase-dependent response to DNA damage in poly(ADP-ribose) polymerase deficient cell lines: basis for a new hypothesis describing the role of poly(ADP-ribose) polymerase in DNA replication and repair

We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl N-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.