GC Transitions Research Articles

Replication-dependent mutagenesis by 5-bromodeoxyuridine: Identification of base change and sequence effects on mutability

The molecular mechanism of reversion induced by 5-bromodeoxyuridine (BrdU) replication-dependent mutagenesis in mammalian cells was studied. Murine cells with single mutant copies of the E. coli gpt gene integrated chromosomally as part of a shuttle vector were mutagenized with BrdU, and GPT+ revertants were selected. Thirteen mutant cell lines (each of which had a gpt gene that varied from the wild-type gene by a different GC----AT base transition in the coding region) were mutagenized, and only four were found to be effectively reverted. All revertant gpt genes that were analyzed had reverted via AT----GC base transition at the original site of mutation, thus demonstrating that replication-dependent mutagenesis by BrdU causes AT----GC transitions. The nine cell lines that were nonrevertible by BrdU replication-dependent mutagenesis could be mutated by this protocol to ouabain resistance as effectively as the four revertible lines, indicating that the nonrevertible lines were susceptible to such mutagenesis. Thus, differences among the cell lines in frequencies of HATr revertants generated by BrdU replication-dependent mutagenesis could not be attributed to differences in general susceptibility of the lines to the mutagenic protocol. The revertible and nonrevertible lines could not be separated according to the position of the original GC----AT transition in the gpt coding region. However, there was evidence that the DNA base sequence flanking the site of mutation affected the susceptibility of that site to BrdU replication-dependent mutagenesis. For example, six of the cell lines tested had gpt genes in which the mutant T residue was immediately adjacent on its 3' side to an A residue, and all six were found to be nonrevertible by BrdU replication-dependent mutagenesis. Furthermore, a target AT base pair flanked by GC base pairs in opposite orientation and either immediately adjacent to or one base removed from the target site on both the 5' and 3' sides appeared to have an increased susceptibility to BrdU replication-dependent mutagenesis.

Relationship between the formation of promutagenic adducts and the activation of the K-ras protooncogene in lung tumors from A/J mice treated with nitrosamines : Belinsky SA, Devereux TR, Maronpot RR, Stoner GD, Anderson MW. Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709. Cancer Res 1989;49:5305-11

Lung and liver tumors were induced in female A/J mice after treatment for 7 weeks (3 times/week, i.p.) with either 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (50 mg/kg) or nitrosodimethylamine (NDMA) (3 mg/kg). Both compounds can be activated via alpha-hydroxylation to methylating agents, while NNK may also undergo hydroxylation at the N-methyl carbon to form a pyridyloxobutylated adduct. The purpose of these studies was to identify and characterize the activated oncogenes present in tumors induced by NDMA and NNK. Following transfection of high molecular weight DNA onto NIH/3T3 mouse fibroblasts, transforming genes were detected in 90% of both NNK- (10 of 11) and NDMA- (9 of 10) induced lung tumors. In contrast, transformation of NIH/3T3 fibroblasts was observed only in 40% (2 of 5) and 13% (1 of 8) of the liver tumors from NNK- and NDMA-treated mice, respectively. Southern blot analysis indicated that the transforming gene present in all lung tumors was an activated K-ras oncogene. Both rearranged bands and amplified signals were detected in the transfectants. The one transformant from the NDMA-induced liver tumor contained an activated K-ras gene. In contrast, the two liver transformants from NNK-induced tumors did not contain an activated ras or raf gene. Hybridization with oligonucleotide probes that were centered around either codon 12 or 61 of the K-ras gene were utilized to localize the mutations. Activation of this gene appeared to occur largely via a mutation in codon 12 (15 of 20 transformants) and was observed with a similar frequency in pulmonary tumors induced by either compound. The remaining mutations were found in codon 61. The specific mutation within these two codons was determined by amplifying the exon containing the base change, followed by direct sequencing. With one exception the mutation observed in codon 12 was a GC to AT transition (GGT to GAT). One transformant contained a GC to TA transversion. The activating mutation detected in codon 61 was always an AT to GC transition of the middle A (CAA to CGA). The GC to AT mutation observed in codon 12 is consistent with the formation of the O6-methylguanine adduct. Similar concentrations (23 to 32 pmol/mumol deoxyguanosine) of this promutagenic adduct were detected in lungs during treatment with either NNK or NDMA.(ABSTRACT TRUNCATED AT 400 WORDS)

Specific Ki‐ras codon 61 mutations may determine the development of urethan‐induced mouse lung adenomas or adenocarcinomas

In A/J strain mice, the carcinogen urethan induces lung adenomas and adenocarcinomas that contain Ki-ras-activating mutations primarily in codon 61. These mutations affect the middle adenine in codon 61 resulting in the substitution of either arginine (AT----GC transition) or leucine (AT----TA transversion) for the wild-type glutamine. To analyze the expression of the wild-type and mutant Ki-ras mRNAs in primary mouse lung tumors and transformed mouse lung cell lines, we utilized reverse transcription of total mRNA and DNA amplification by the polymerase chain reaction. The wild-type allele of codon 61 was expressed in all normal lung and primary tumor samples and in all transformed cell lines, except one. Significantly, the leucine-substituted allele was expressed primarily in very small lung adenomas, whereas the arginine-substituted allele was expressed in large lung adenocarcinomas and transformed lung cell lines. The relative amounts of expression of the mutant versus wild-type Ki-ras alleles and the total Ki-ras mRNA expression was similar in both lung adenomas and adenocarcinomas. Further, the arginine mutant allele was present in adenocarcinomas having either alveolar or papillary tumor morphologies. These results suggest that the specific activating Ki-ras mutation is more critical to either lung adenoma or adenocarcinoma development than is the tumor's cell of origin or the extent to which the mutant alleles are expressed. A distinct role of the specific activating Ki-ras mutations in affecting lung tumor growth or malignant potential is indicated.

Formaldehyde-induced and spontaneous alterations in human hprt DNA sequence and mRNA expression

Human lymphoblast mutants at the X-linked hprt locus have been examined by Southern blot, Northern blot and DNA sequence analysis. A previous study had shown that approximately a third of the spontaneously-arising mutants and half those induced by formaldehyde showed no alteration in restriction fragment pattern and thus were classified as point mutationns. In this report, Northern blot analysis was used to show that these point mutants fall into 4 categories; normal size and amount of RNA, normal size but reduced amounts, reduced size of RNA or no RNA. Sequence analyses of cDNAs prepared from hprt mRNAs were performed on 1 spontaneous and 7 formaldehyde-induced mutants with normal Northern blots. The spontaneous mutant was caued by an AT→GC transition. 6 of the formaldehyde-induced mutants were base substitutions, all of which occurred at AT base-pairs. There was an apparent hot spot, in that 4 6 independent mutants were AT→CG transversions at one specific site. The remaining mutant had lost exon 8.

Mutational spectrum analysis of umuC-independent and umuC-dependent γ-radiation mutagenesis in Escherichia coli

gamma-Radiation mutagenesis (oxic versus anoxic) was examined in wild-type, umuC and recA strains of Escherichia coli K-12. Mutagenesis [argE3(Oc)----Arg+] was blocked in a delta (recA-srlR)306 strain at the same doses that induced mutations in umuC122::Tn5 and wild-type strains, indicating that both umuC-independent and umuC-dependent mechanisms function within recA-dependent misrepair. Analyses of various suppressor and back mutations that result in argE3 and hisG4 ochre reversion and an analysis of trpE9777 (+1 frameshift) reversion were performed on umuC and wild-type cells irradiated in the presence and absence of oxygen. While the umuC strain showed the gamma-radiation induction of base substitution and frameshifts when irradiated in the absence of oxygen, the umuC mutation blocked all oxygen-dependent base-substitution mutagenesis, but not all oxygen-dependent frameshift mutagenesis. For anoxically irradiated cells, the yields of GC----AT [i.e., at the supB and supE (Oc) loci] and AT----GC transitions (i.e., at the argE3 and hisG4 loci) were essentially umuC independent, while the yields of (AT or GC)----TA transversions (i.e., at the supC, supL, supM, supN and supX loci) were heavily umuC dependent. These data suggest new concepts about the nature of the DNA lesions and the mutagenic mechanisms that lead to gamma-radiation mutagenesis.

Mutational specificities of 1′-acetoxysafrole, N-benzoyloxy- N-methyl-4-aminoazobenzene, and ethyl methanesulfonate in human cells

We have used an oriP-tk shuttle vector t determine the types of mutations induced in human cells by ethyl methanesulfonate (EMS), 1'-acetoxysafrole (AcOS), and N-benzoyloxy- N-methyl-4-aminoazobenzene (BzOMAB). Plasmid DNA was treated in vitro with mutagen and electroporated into human lymphoblastoid cells. After replication of the vector in human cells, plasmids were analyzed for mutations in the herpes simplex virus type 1 thymidine kinase gene. Ethyl methanesulfonate induced predominantly GC → AT transition mutations. Treatment of the shuttle vector with AcOS induced 5 of the 6 possible base substitution mutations, including GC → AT (32%) and AT → GC (14%) transition mutations, GC → TA (%), GC → CG (18%), and AT → TA (14%) transversion mutations, as well as a low frequency (9%) of −1 frameshift mutations at GC base pairs. Replication in human cells of DNA modified with BzOMAB yielded a significant increase (17-fold) in the frequency of deletion mutations relative to solvent-treated DNA. A majority (94%) of the point mutations induced by BzOMAB occurred at GC base pairs and were predomianntly GC → AT transitions (33%) and −1 frameshift (22%) mutations, with the remainder consisting mainly of transversions at GC base pairs (28%). The broad spectrum of base substitution mutations observed for AcOS and BzOMAB may indicate the frequent insertion of a variety of bases during replicative bypass of aralkylated bases in human cells.

Mutation spectra of N-ethyl-N'-nitro-N-nitrosoguanidine and 1-(2-hydroxyethyl)-1-nitrosourea in Escherichia coli.

DNA sequencing was used to determine the specific types of DNA base changes induced following in vivo exposure of Escherichia coli to the ethylating agent N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and the hydroxyethylating agent 1-(2-hydroxyethyl)-1-nitrosourea (HENU) using the xanthine guanine phosphoribosyltransferase (gpt) gene as the genetic target. We observed that 22/30 of the ENNG-induced mutations were GC----AT transitions, 4/30 were AT----GC transitions, 3/30 were AT----TA transversions, and 1/30 was an AT----CG transversion. We observed that 37/40 HENU-induced mutations were GC----AT transitions and that the remaining 3/40 were AT----GC transitions. A majority of the GC----AT transitions induced by ENNG and HENU (68% and 73%, respectively) occurred at the second guanine of the sequence 5'-GG(A or T)-3'; this sequence specificity was similar to that previously seen with the alkylating agents N-methyl- and N-ethyl-N-nitrosourea (MNU and ENU) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). A DNA strand preference for the G----A changes (antisense strand), previously noted for MNU, ENU, and MNNG, was observed following exposure to HENU and ENNG. The AT----GC transitions induced by ENNG, HENU, and ENU also exhibit a sequence specificity with 13/13 mutations occurring at the T of the sequence 5'-NTC-3'. A strand preference was not apparent for these mutations.

An analysis of the mutagenicity of 1,2-dibromoethane to Escherichia coli: Influence of DNA repair activities and metabolic pathways

The mutagenicity of 1,2-dibromoethane (EDB) to Escherichia coli was reduced by the UV light-induced excision repair system but unaffected by the loss of a major apurinic/apyrimidinic site repair function. At high doses, 70–90% of the EDB-induced mutations were independent of SOS-mutagenic processing and approximately 50% were independent of glutathione conjugation. The SOS-independent mutations induced by EDB were unaffected by the enzymes that repair alkylation-induced DNA lesions. EDB-induced base substitutions were dominated by GC to AT and AT to GC transitions. These results suggest that EDB-induced premutagenic lesions have some, but not all, of the characteristics of simple alkyl lesions.

Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

The role of deoxyribonucleotide metabolism in 5-bromo-2′-deoxyuridine mutagenesis in mammalian cells

The effects of deoxyribonucleoside triphosphate (dNTP) pool imbalance on the induction of mutations and siste-chromatid exchanges (SCEs) by 5-bromo-2′-deoxyuridine (BrdUrd) in mammalian cells is reviewed. The INC BrdUrd mutagenesis protocol involves the incorporation of BrdUrd into DNA under conditions of specific dNTP pool imbalance, while the REP BrdUrd mutagenesis protocol involves the replication of 5-bromouracil (BrUra)-substituted DNA in the presence of specific (but different) dNTP pool imbalance. Biochemical and genetic analyses of both the INC and REP mutagenesis protocols provided evidence that (1) INC mutagenesis resulted from errors of incorporation due to the mispairing of BrdUTP with a guanine residue in replicating DNA leading to GC to AT transitions and (2) REP mutagenesis resulted from errors of replication due to the mispairing of dGTP with a BrURA residue in replicating DNA leading to AT to GC transitions. Further analyses involving different cell lines has led to an hypothesis describing the role of mismatch repair in the induction of mutations and SCEs.

Molecular analysis of mutations induced in human cells by N-ethyl-N-nitrosourea.

Mutational activation of cellular proto-oncogenes is an important event in the pathogenesis of chemically induced tumors. We have used the ori P-tk shuttle vector, pHET, to analyze the types of DNA sequence changes induced after treating mammalian cells with the carcinogen N-ethyl-N-nitrosourea (ENU). This shuttle vector contains the putative replication origin of the Epstein-Barr virus (EBV) and is stably maintained as a plasmid in EBV-transformed human lymphoblastoid cells. Populations of plasmid-bearing cells were treated with ENU, and plasmid DNA was isolated approximately 7-8 population doublings after treatment for analysis of mutations induced at the herpes simplex virus type 1 thymidine kinase (HSV-tk) target gene. After ENU treatment, frequencies of four of the six possible base substitution mutations significantly increased. Transition mutations were the most common sequence change: 48% of the 46 mutants sequenced were GC----AT transitions and 17% were AT----GC transitions. In addition, the number of AT----TA (20%) and AT----CG (9%) transversion mutations significantly increased after ENU treatment. Based on the comparison of mutations induced by ENU in human cells with the types of base pair changes previously reported for other alkylating agents, we propose that the O2-ethylthymine adduct may be a significant premutagenic lesion in mammalian cells, capable of resulting in AT base pair transversion mutations. Studies from other laboratories have demonstrated the importance of AT----TA transversion mutations in the activation of cellular proto-oncogenes by ENU.

Different mutational profiles induced by N-nitroso-N-ethylurea: effects of dose and error-prone DNA repair and correlations with DNA adducts.

The DNA adducts and mutational profile produced by N-nitroso-N-ethylurea (ENU) in Salmonella are examined. The adduct profile produced by ENU in isolated DNA and at two doses in Salmonella were similar, with one exception: O6-ethylguanine (O6-EtG) was not detected at the low dose in Salmonella. This adduct was presumably repaired by a constitutive repair system. The premutagenic adducts, O2-ethyl-thymidine (O2-EtdT) and O4-ethylthymidine (O4-EtdT) were detected, with the former adduct present at higher levels. The mutational profile was also determined at the same doses by utilizing a system involving a series of histidine auxotrophs of Salmonella with differing mutagenic specificities and a further subclassification of the revertants. Four different patterns of mutagenesis were observed; these were dependent on dose and on the presence or absence of the plasmid pKM101. The mutational spectrum produced at the higher dose in strains without the plasmid consisted mainly of GC----AT transitions. At the high dose, in strains harboring pKM101, three base changes contributed importantly to the mutational spectrum: GC----AT, AT----GC, and AT----CG. At the low dose in the strains without pKM101, little mutagenesis was observed, and in strains containing pKM101, mutagenesis was greatly enhanced with the most frequent mutations resulting from AT----GC and AT----CG base changes. O6-EtG was presumably responsible for the bulk of the GC----AT transitions at the high dose. Calculations and evidence are presented indicating that O2-EtdT is responsible for at least some of the mutagenesis that occurs at AT base pairs. O4-EtdT and O2-EtdT are probably responsible for a major fraction of the AT----GC transitions, and we suggest that error-prone repair activity acting on O2-EtdT and/or O4-EtdT results in the AT----CG transversions.

Sequence specificity of streptozotocin-induced mutations

The isolation and characterization of streptozotocin (STZ)-induced mutations in the phage P22 mnt repressor gene is described. Cells carrying the plasmid-borne mnt gene were exposed to STZ to give 10-20 percent survival and at least an eleven-fold increase in mutation frequency. DNA sequence analysis showed that 50 of 51 STZ-induced mutations were GC to AT transitions, and one was an AT to GC transition. We have also compared the STZ mutational spectrum to that for N-methyl-N'-nitro-N-nitroso-guanidine (MNNG). There are sites in the mnt gene which are mutated only by STZ; only by MNNG, or by both agents. Sites at which only STZ induced GC to AT transition mutations occur were in sequences that are pyrimidine rich 5' to the mutated site and purine rich 3' to the mutated site. Induction of mutations by both STZ and MNNG should be considered to maximize the number of mutable sites.

Conditional mutations ofSalmonella typhimuriumthat are suppressed by anaerobic or aerobic environments

Mutant strains of Salmonella typhimurium were isolated that grew on a minimal medium only in an aerobic environment or only in an anaerobic environment. Mutants that grew only anaerobically were induced by mutagens such as deoxybromouridine and deoxyiodouridine that promote AT → GC transitions whereas those that grew only aerobically were induced by mutagens such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) that promote GC → AT transitions. This mutagen selectivity and the ability of mutant strains to grow on minimal medium in one environment suggested that the changes in DNA occurred outside the coding regions of genes. These results provide a means to investigate the mechanisms by which organisms adapt to, or are restricted by, their environment.

Conditional mutations of Salmonella typhimurium that are suppressed by anaerobic or aerobic environments

Mutant strains of Salmonella typhimurium were isolated that grew on a minimal medium only in an aerobic environment or only in an anaerobic environment. Mutants that grew only anaerobically were induced by mutagens such as deoxybromouridine and deoxyiodouridine that promote AT → GC transitions whereas those that grew only aerobically were induced by mutagens such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) that promote GC → AT transitions. This mutagen selectivity and the ability of mutant strains to grow on minimal medium in one environment suggested that the changes in DNA occurred outside the coding regions of genes. These results provide a means to investigate the mechanisms by which organisms adapt to, or are restricted by, their environment.

Transversion-specific purine analogue mutagens and the mechanism of hydroxylaminopurine mutagenesis

The tryptophan synthetase gene A series of mutants in E. coli has been used to examine the mutational specificity of over 80 purine base analogues. (1) 4 purine analogues have been discovered that solely cause transversions. (2) Evidence is presented that hydroxylaminopurine mutagenesis is caused by a covalent reaction of these compounds with DNA. The transversion-causing purine analogues are derivatives of 2-aminopurine (2AP) and 2,6-di-aminopurine (2,6DAP). They stimulate the full reversion frequency of those trp A which can revert through an AT → CG transversion. 8 purine base analogues have been found that induce the AT → CG transversion at the trp A88 site; and 2-amino-6-methylaminopurine (2A6MAP) stimulates by 124-fold, 2-amino-6-ethylaminopurine by 20-fold, 2-methylaminopurine (2MAP) by 9.4-fold, 2,6-bismethylamino-purine by 25-fold, 2AP by 230-fold, 2,6DAP by 15-fold, 2.6-diaminopurine riboside by 5-fold, and 2-hydroxylaminopurine by 11-fold. The last 4 analogues also cause transitions. 2A6MAP, 2-amino-6-ethyl-aminopurine and 2,6-bismethylaminopurine stimulate only the AT → CG transversion while 2MAP additionally gives rise to AT ↔ TA transversions. By testing other negative 2AP derivatives, the structural requirements necessary for AT → CG transversion mutagenesis have been determined. All 12 hydroxylaminopurine base analogues tested, 2,6-dimethoxyaminopurine and 2-hydrazinopurine were found to cause transition mutations. All of the compounds stimulated the AT → GC transition (by up to 1000-fold) and 11 of the 14 base analogues raised the GT → AT transition (by up to 450-fold). On increasing the hydroxylaminopurine concentration, the mutation frequency also increased concomitantly. Since 6-hydroxylamino-9-methylpurine and 6-methylhydroxylaminopurine cause transitions, the mechanism of hydroxylaminopurine mutagenesis cannot be entirely due to an alteration in tautomeric equilibria or “wobble” type base mispairing. It is proposed that a major mechanism for hydroxylaminopurine mutagenesis is due to the reaction of these compounds with the O 6-position of guanine and the O 4-position of thymine.

O6-Methylguanine specifically induces AT→GC transition mutations

O6-Methylguanine specifically induces AT→GC transition mutations

A single-base change within the DNA polymerase locus of herpes simplex virus type 2 can confer resistance to aphidicolin.

An aphidicolin-resistant (Aphr) mutant of herpes simplex virus (HSV) type 2 strain 186 previously has been shown to induce an altered viral DNA polymerase that is more resistant to aphidicolin and more sensitive to phosphonoacetic acid (PAA) than is wild-type DNA polymerase. In this study the mutation responsible for the aphidicolin-resistant phenotype was physically mapped by marker transfer experiments. The physical map limits for the Aphr mutation were contained in a 1.1-kilobase pair region within the HSV DNA polymerase locus. The 1.1-kilobase-pair fragment of the Aphr mutant also conferred hypersensitivity to PAA, and DNA sequence analysis revealed an AT to GC transition within this fragment of the Aphr mutant. Analysis of the three potential open reading frames within the 1,147-base-pair fragment and comparison with the amino acid sequence of DNA polymerase of HSV type 1 indicated that the Aphr mutant polymerase had an amino acid substitution from a tyrosine to a histidine in the well-conserved region of the DNA polymerase. These results indicate that this single amino acid change can confer altered sensitivity to aphidicolin and PAA and suggest that this region may form a domain that contains the binding sites for substrates, PPi, and aphidicolin.

Mutation spectrum inEscherichia coliDNA mismatch repair deficient (mutH) strain

The Dam-directed post-replicative mismatch repair system of Escherichia coli removes base pair mismatches from DNA. The products of the mutH, mutL and mutS genes, among others, are required for efficient mismatch repair. Absence of any of these gene products leads to persistence of mismatches in DNA with a resultant increase in spontaneous mutation rate. To determine the specificity of the mismatch repair system in vivo we have isolated and characterized 47 independent mutations from a mutH strain in the plasmid borne mnt repressor gene. The major class of mutations comprises AT to GC transitions that occur within six base pairs of the only two 5'-GATC-3' sequences in the mnt gene. In the wild type control strain, insertion of the IS1 element was the major spontaneous mutational event. A prediction of the Dam-directed mismatch repair model, that the mutation spectra of dam and mutH strains should be the same, was confirmed.