Hotspot Sites Research Articles

Intramolecular Higher Order Packing of Parallel Quadruplexes Comprising a G:G:G:G Tetrad and a G(:A):G(:A):G(:A):G Heptad of GGA Triplet Repeat DNA

GGA triplet repeats are widely dispersed throughout eukaryotic genomes and are frequently located within biologically important regions such as gene regulatory regions and recombination hot spot sites. We determined the structure of d(GGA)4 (12-mer) under physiological conditions and founded the formation of an intramolecular parallel quadruplex for the first time. Later, a similar architecture to that of the intramolecular parallel quadruplex was found for a telomere DNA in the crystalline state. Here, we have determined the structure of d(GGA)8 (24-mer) under physiological conditions. Two intramolecular parallel quadruplexes comprising a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad are formed in d(GGA)8. These quadruplexes are packed in a tail-to-tail manner. This is the first demonstration of the intramolecular higher order packing of quadruplexes at atomic resolution. K+ ions, but not Na+ ones, are critically required for the formation of this unique structure. The elucidated structure suggests the mechanisms underlying the biological events related to the GGA triplet repeat. Furthermore, in the light of the structure, the mode of the higher order packing of the telomere DNA is discussed.

Spontaneous Hotspot Mutations Resistant to Mismatch Correction in Escherichia coli: Transcription-dependent Mutagenesis Involving Template-switching Mechanisms

The generation and stabilization of spontaneous mutations are affected by many factors, including the accuracy of DNA replication, the generation of spontaneous DNA lesions, and the capacity of mutation-avoidance systems. However, little is known about the causes of spontaneous mutations in cells with fully active mutation-avoidance systems. Using the rpsL forward mutation assay, we previously found that the directionality of replication fork movement significantly affects spontaneous mutagenesis in Escherichia coli. In particular, sequence substitutions and a hotspot type of single-base frameshift, both of which are caused by quasipalindrome-directed mutagenesis, appeared to depend on the directionality of the replication fork. These mutations are also resistant to post-replicative mismatch correction. Here, we show that the level of transcription of the rpsL gene strongly affects spontaneous mutagenesis at two mutational hotspot sites in the target sequence, one for a T→G base substitution and the other for a+1 single-base frameshift. Mutation frequencies at the hotspot sites were below a detectable level when the transcription of the target sequence was tightly suppressed, but were dramatically increased when the target sequence was highly transcribed. Both of the hotspot mutations were also dependent on the directionality of the replication fork and were caused by quasipalindrome-directed mutagenesis. The frequencies of the hotspot mutations were unchanged in a mismatch-repair deficient strain, indicating that the hotspot mutations are resistant to the mismatch correction. Based on these findings, we propose a novel mutagenic process for these hotspot mutations that depends on transcription and involves template-switching mechanisms induced by spontaneous DNA lesions.

An in Vivo Approach to Identifying Sequence Context of 8-Oxoguanine Mutagenesis

Base substitution mutations are not distributed randomly in that most are located at a few specific hotspots sites. We have been studying 7,8-dihydro-8-oxoguanine mutagenesis in Escherichia coli in the supF gene carried in a plasmid. Among hotspots, guanine within the 5′-AGA-3′ located in the anticodon site was susceptible to the induction of G:C→T:A transversion. In this study, we constructed variants of the supF gene in which the hotspot 5′-AGA-3′ was modified to 5′-AGT-3′, 5′-AGG-3′ and 5′-AGC-3′ to determine the influence of 3′ neighboring base on G:C→T:A mutational activity. Using these variant supF genes propagated in a 7,8-dihydro-8-oxoguanine repair-deficient host, we found that guanine within 5′-AGA-3′ and 5′-AGG-3′ produce G:C→T:A, but guanine within 5′-AGT-3′ and 5′-AGC-3′ reduce the formation of G:C→T:A. These changes were thus due to the effect of sequence context on the efficiency of mutation formation at the sites of 7,8-dihydro-8-oxoguanine. We also observed a longer range base-pair effect on hotspot formation.

Role of DNA ligase in the illegitimate recombination that generates lambdabio-transducing phages in Escherichia coli.

We studied the role of DNA ligase in illegitimate recombination in Escherichia coli. A temperature-sensitive mutation in the lig gene reduced the frequency with which lambdabio-transducing phages were generated to 10-14% of that of wild type under UV irradiation. Reintroduction of the lig gene into this mutant restored the frequency of recombinant phage generation to that of wild type. Furthermore, overexpression of DNA ligase enhanced illegitimate recombination by 10-fold with or without UV irradiation. In addition, when DNA ligase was present in only limited amounts, UV-induced or spontaneous illegitimate recombination occurred exclusively at hotspot sites that have relatively long sequences of homology (9 or 13 bp). However, when DNA ligase was overexpressed, most of the illegitimate recombination took place at non-hotspot sites having only short sequences of homology (<4 bp). Thus, the level of ligase activity affects the frequency of illegitimate recombination, the length of sequence homology at the recombination sites, and the preference for recombination at hotspots, at least after UV irradiation. These observations support our hypothesis that the illegitimate recombination that generates lambdabio-transducing phages is mediated by the DNA break-and-join mechanism.

Directionality of DNA replication fork movement strongly affects the generation of spontaneous mutations in Escherichia coli

Using a pair of plasmids carrying the rpsL target sequence in different orientations to the replication origin, we analyzed a large number of forward mutations generated in wild-type and mismatch-repair deficient (MMR−) Escherichia coli cells to assess the effects of directionality of replication-fork movement on spontaneous mutagenesis and the generation of replication error. All classes of the mutations found in wild-type cells but not MMR− cells were strongly affected by the directionality of replication fork movement. It also appeared that the directionality of replication-fork movement governs the directionality of sequence substitution mutagenesis, which occurred in wild-type cells at a frequency comparable to base substitutions and single-base frameshift mutations. A very strong orientation-dependent hot-spot site for single-base frameshift mutations was discovered and demonstrated to be caused by the same process involved in sequence substitution mutagenesis. It is surprising that dnaE173, a potent mutator mutation specific for sequence substitution as well as single-base frameshift, did not enhance the frequency of the hot-spot frameshift mutation. Furthermore, the frequency of the hot-spot frameshift mutation was unchanged in the MMR− strain, whereas the mutHLS-dependent mismatch repair system efficiently suppressed the generation of single-base frameshift mutations. These results suggested that the hot-spot frameshift mutagenesis might be initiated at a particular location containing a DNA lesion, and thereby produce a premutagenic replication intermediate resistant to MMR. Significant numbers of spontaneous single-base frameshift mutations are probably caused by similar mechanisms.

New comprehensive denaturing‐gradient‐gel‐ electrophoresis assay for KRAS mutation detection applied to paraffin‐embedded tumours

A comprehensive mutation detection assay is presented for the entire coding region and all splice site junctions of the KRAS oncogene. The assay is based on denaturing gradient gel electrophoresis and applicable to archival paraffin-embedded tumour material. All KRAS amplicons are analysed within two lanes of a DGGE gel under a single set of experimental conditions. Six known codon 12 mutations in genomic DNA from different paraffin-embedded tumours could readily be detected. When testing 35 paraffin-embedded Dukes' C colorectal carcinomas for unknown mutations, 12 tumours were found with mutations in codons 12 or 13. None of the tumours appeared to have a codon 61 mutation. In nine tumours, however, 11 additional sequence variations were found outside the hot-spot codons. None of these occurred in germline DNA from 57 individuals. Of these variations, three are considered as significant mutations, as they result in a non-conservative substitution of amino acid residues essential for the functioning of the KRAS protein. Thus, in total, 15 of the 35 Dukes' C tumours (43%) had a KRAS mutation of functional significance. Moreover, a novel exon 4B polymorphism was found to occur in 10 of the 35 tumours. The results of this study suggest that in restricting analysis of KRAS to hot-spot mutation sites only, significant information may be missed. © 2000 Wiley-Liss, Inc.

Unrepaired fjord region polycyclic aromatic hydrocarbon-DNA adducts in ras codon 61 mutational hot spots.

The fjord region diol-epoxide metabolites of polycyclic aromatic hydrocarbons display stronger tumorigenic activities in rodent studies than comparable bay region diol-epoxides, but the molecular basis for this difference between fjord and bay region derivatives is not understood. Here we tested whether the variable effects of these genotoxic metabolites of polycyclic aromatic hydrocarbons may result from different DNA repair reactions. In particular, we compared the repairability of DNA adducts formed by bay region benzo[a]pyrene (B[a]P) diol-epoxides and the structurally similar but significantly more tumorigenic fjord region diol-epoxide metabolites of benzo[c]phenanthrene (B[c]Ph). For that purpose, we incorporated both types of polycyclic aromatic hydrocarbon adducts into known hot spot sites for carcinogen-induced proto-oncogene activation. Synthetic DNA substrates were assembled using a portion of human N-ras or H-ras that includes codon 61, and stereospecific B[a]P or B[c]Ph adducts were synthesized on adenine N6 at the second position of these two ras codon 61 sequences. DNA repair was determined by incubating the site-directed substrates in human cell extracts, followed by electrophoretic visualization of radiolabeled oligonucleotide excision products. These cell-free assays showed that all tested bay region B[a]P-N6-dA adducts are removed by the human nucleotide excision repair system, although excision efficiency varied with the particular stereochemical configuration of each B[a]P residue. In contrast, all fjord region B[c]Ph-N6-dA adducts located in the identical sequence context and with exactly the same stereochemical properties as the corresponding B[a]P lesions were refractory to the nucleotide excision repair process. These findings indicate that the exceptional tumorigenic potency of B[c]Ph or related fjord region diol-epoxides may be attributed, at least in part, to slow repair of the stable base adducts deriving from the reaction of these compounds with DNA.

New comprehensive denaturing-gradient-gel- electrophoresis assay forKRAS mutation detection applied to paraffin-embedded tumours

A comprehensive mutation detection assay is presented for the entire coding region and all splice site junctions of the KRAS oncogene. The assay is based on denaturing gradient gel electrophoresis and applicable to archival paraffin-embedded tumour material. All KRAS amplicons are analysed within two lanes of a DGGE gel under a single set of experimental conditions. Six known codon 12 mutations in genomic DNA from different paraffin-embedded tumours could readily be detected. When testing 35 paraffin-embedded Dukes' C colorectal carcinomas for unknown mutations, 12 tumours were found with mutations in codons 12 or 13. None of the tumours appeared to have a codon 61 mutation. In nine tumours, however, 11 additional sequence variations were found outside the hot-spot codons. None of these occurred in germline DNA from 57 individuals. Of these variations, three are considered as significant mutations, as they result in a non-conservative substitution of amino acid residues essential for the functioning of the KRAS protein. Thus, in total, 15 of the 35 Dukes' C tumours (43%) had a KRAS mutation of functional significance. Moreover, a novel exon 4B polymorphism was found to occur in 10 of the 35 tumours. The results of this study suggest that in restricting analysis of KRAS to hot-spot mutation sites only, significant information may be missed.

Development of the hot spot–combined PCR assay for detection of retroviral insertions into Marek’s disease virus

A two-step PCR, the Hot Spot–combined PCR assay, was developed for the identification and characterization of recombinant viruses in Marek’s disease (herpes) and retrovirus co-infections. In the first PCR the herpesvirus genomic fragment, that was recognized in previous studies as a hot spot site for retroviral integration was amplified [reviewed in Bronovskis, P., Kung, H.-J., 1996. Retrotransposition and herpesvirus evolution. Virus Genes 11, 259–270]. The products served for a second amplification step, performed in six PCR sets, using the six possible combinations of the two herpes and the retrovirus primer sets. Development of the assay employed DNA of the recombinant virus, RM1, which was created by in vitro co-cultivation of Marek’s disease and reticuloendotheliosis viruses [Isfort et al., 1992. Proc. Natl. Acad. Sci. 89, 991–995; Witter et al., 1997. Avian Dis. 41, 407–421]. As the retroviral insertion site and junction sequences were determined previously [Jones et al., 1996. J. Virol. 70, 2460–2467], RM1 served in the present study as a test virus for the development of the new assay. It is shown now that the Hot Spot-combined PCR can detect the retroviral insert in RM1, the MDV integration site and the insert orientation. For confirmation the herpes and retrovirus chimeric PCR products were sequenced and the results were similar to those published previously [Jones et al., 1996. J. Virol. 70, 2460–2467]. This assay might be adopted in additional systems to detect foreign inserts at suspected genomic sites.

On the spontaneous mutability of CpG sites in cultured S49 mouse lymphoma cells.

The frequent occurrence of human mutations at CpG dinucleotide sites has been attributed to cytosine methylation and hydrolytic deamination of the resulting 5-methylcytosine residue. Previously, we reported an unusually strong hotspot for spontaneous transitions at a CpG site in the gene for regulatory (R) subunit of protein kinase A in S49 mouse lymphoma cells. Now, using polymerase chain reaction-based methods to screen mutant populations for mutations at particular CpG sites, we show that two methylated CpG sites in the gene for hypoxanthine phosphoribosyltransferase are much less mutable than the R subunit hotspot site, suggesting that different methylated CpG sites are differentially susceptible to spontaneous mutation. We also present data on spontaneous R subunit mutations in cloned populations of 5-azacytidine-treated S49 cells that had been demethylated at the hotspot site in both R subunit alleles. Of 13 independent mutants isolated from populations grown from fully demethylated cells, seven had the hotspot mutation. We conclude that CG-->TA mutations at strong CpG hotspots do not require prior methylation of CpG sites.

Integrated physical and genetic mapping of Bacillus cereus and other gram-positive bacteria based on IS231A transposition vectors.

The genome structure of Bacillus cereus is relatively complex, its DNA being modulated between a size-varying chromosome and large plasmids. To study the genetic organization of the B. cereus type strain ATCC 14579, thermosensitive transposition vectors were designed on the basis of IS231A-derived cassettes containing uncommon restriction sites. A highly preferred insertion site for IS231A was detected in the chromosome by Southern blotting and pulsed-field gel electrophoresis (PFGE) analyses of independent insertion mutants. However, once this insertional hot spot was occupied, secondary IS231A insertions occurred randomly, as demonstrated by isolation of independent B. cereus auxotrophs at a frequency of approximately 0.6%. The hot-spot site, as well as several auxotrophic mutations, were mapped by using NotI, SfiI, and AscI PFGE restriction profiles. It was confirmed by sequencing that one of the insertions, generating an Ade- phenotype, had disrupted a gene of the purine synthesis pathway. These results showed that combined PFGE and sequencing analyses of mini-IS231A insertions enable the construction of integrated physical and genetic maps of B. cereus type strain. Moreover, the presence of the ultrarare I-SceI restriction site in the mini-IS231A allowed the isolation, in double-insertion mutants, of contiguous and nonoverlapping large chromosomal fragments, convenient for direct sequencing. The system detailed in this report is therefore a powerful tool for comparative genetic studies among members of the B. cereus group (i.e., B. cereus, B. thuringiensis, B. mycoides, and B. anthracis) and could also be applied to more distantly related gram-positive bacteria.

Suppressors of cleavage-site mutations in the p62 envelope protein of Semliki Forest virus reveal dynamics in spike structure and function.

The E2 spike glycoprotein of Semliki Forest virus is produced as a p62 precursor protein, which is cleaved by host proteases to its mature form, E2. Cleavage is not necessary for particle formation or release but is necessary for infectivity. Previous results had shown that phenotypic revertants of cleavage-deficient p62 mutants are generated, and here we show that these may contain second-site suppressor mutations in the vicinity of the cleavage site. These hot-spot sites were mutated to abolish the generation of such suppressor mutations; however, secondary mutations in another distant domain of the E2 protein appeared instead, all of which still caused cleavage-deficient mutations. Such mutants grew very poorly and were inefficient in virus entry and release. The mutated sites define domains of the spike protein which probably interact to regulate its structure and function. Because of their highly attenuated phenotype and the lower probability of reversion, the new mutations close to the cleavage site were used to make new helper vectors for packaging of recombinant RNA into infectious particles, thus increasing further the biosafety of the vector system based on the Semliki Forest virus replicon.

Localization of the (TTAGGG)n telomeric sequence in zebrafish chromosomes

The distribution of the (TTAGGG)n telomeric sequence in Danio rerio (zebrafish) metaphase chromosomes was studied by fluorescence in situ hybridization. This study continues the analysis of the telomeric sequence distribution in fish and confirms that the (TTAGGG)n sequence is conserved in the telomeres of this group of vertebrates. The hybridization signals were restricted to telomeres in all the individuals analysed. With the degree of resolution currently available, no interstitial sites of the telomeric sequence were detected, which suggests that this type of hot-spot recombination site might be absent in zebrafish chromosomes.

Spectra of spontaneous mutations at the hprt locus in colorectal carcinoma cell lines defective in mismatch repair.

Spectra of spontaneous mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in colon carcinoma cell lines HCT116 and HCT-15 deficient in mismatch repair and displaying mutator phenotypes were determined. HCT116 and HCT-15 cells, respectively, harbour a mutation in the mismatch repair gene hMLH1 and GTBP. The mutation frequency at the hprt locus in both cell lines was elevated by about two orders, but the microsatellite instability in HCT116 cells was one order higher than in HCT-15 cells. Except for one mutant of HCT-15, all the mutations (114/115) were point mutations; base substitutions of various types and frameshifts (deletions/insertions of less than a few bases, predominantly of +/-1 bp). Base substitutions (57%) and frameshifts (43%) occurred at a comparable rate in HCT116, whereas base substitutions (92%) were the major mutational events in HCT-15. Most frameshifts in HCT116 occurred at sites of monotonous or short tandem repeating sequences, and two of these sites, where there was a run of six Gs and four As, were hot spots. Three hot spot sites of base substitutions were found in HCT-15; two of them at splice acceptor sites, the other at the CpG site shared with HCT116. The distinct mutation spectra of the HCT116 and HCT-15 cell lines may reflect functional differences in the hMLH1 and GTBP gene products in mismatch repair. The gene product GTBP may be involved in the preferential repair of base mismatches, and MLH1 in the repair of both base mismatches and deletions/insertions of less than a few bases. These results suggest that mismatch repair deficiency affects the microsatellite stability as widely reported in colorectal tumour cells, but that it may not severely affect chromosome integrity as the karyotypes of these tumour cells are, unlike other tumour cells, relatively stable.

RecQ DNA helicase is a suppressor of illegitimate recombination in Escherichia coli.

Bloom syndrome and Werner syndrome are genetic disorders in which an increased rate of chromosomal abnormality is observed. The genes responsible for these diseases, BLM and WRN, have been cloned and identified as homologs of the Escherichia coli recQ genes. We studied the effect of recQ mutations on illegitimate recombination, which is an aberrant biological event related to the chromosomal abnormality in humans, and found that a variety of recQ mutations increased spontaneous illegitimate recombination by 20- to 300-fold and increased UV light-induced illegitimate recombination by 10- to 100-fold. Most lambda bio or lambda pro transducing phages are formed by the recombination events at several hot spots, which are enhanced by the recQ mutation. The analysis of nucleotide sequences at the recombination junction in the transducing phages indicates that recombination at the hot spot sites as well as the non-hot spot sites takes place between short homologous sequences. Enhancement of the recombination in the recQ mutants also occurs in the recA, recBC sbcBC, or recBC sbcA backgrounds, indicating that these recombination events are mediated by none of the known recombination pathways, RecBC, RecF, and RecE. We therefore concluded that the RecQ function suppresses illegitimate recombination that depends on short homologous regions. We discuss a model, based on the 3'-to-5' helicase activity of RecQ, to explain the role of this protein as a suppressor of illegitimate recombination.

Mutational specificity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in Escherichia coli: comparison of in vivo with in vitro exposure of the supF gene.

Forward mutations induced by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in the supF gene of Escherichia coli were recovered from bacteria deficient in nucleotide excision repair and in DNA-alkyltransferase activity. Bacteria were exposed to 0.4 mM CCNU (in vivo supF mutagenesis), increasing the overall mutation frequency 15.7-fold above the spontaneous value. A total of 73 independent supF- mutants were sequenced. The resulting mutation spectrum was compared with those obtained in bacteria and mammalian cells following the classical shuttle-vector approach (in vitro supF mutagenesis). In vivo CCNU mutagenesis in E. coli yielded a large number of deletions (20/73), in agreement with mammalian data but distinct from in vitro bacterial spectra, which are almost exclusively composed of G:C-->A:T transitions. A substantial proportion (6/18) of CCNU-induced deletions (> 3 bp) involved repeated DNA sequences, suggesting a contribution of a slippage-misalignment process in the generation of this mutation class. Substitutions occurred primarily at G:C base pairs (44/53) and were predominantly G:C-->A:T transitions (39/53). This mutational change was attributed to the mispair potential of the O6-chloroethylguanine lesion with thymine. Most G:C-->A:T transitions (34/39) were located at three 5'-GG-3' hotspot sites (positions 123, 160, and 168). The distribution of hotspot sites for G:C-->A:T substitutions differed as a function of the in vivo or in vitro chemical modification of the supF-bearing plasmids and revealed significant differences in the DNA strand distribution of this mutational event. Our data suggest that the transcriptional status of the target gene has strong influence on the probability of O6-chloroethylguanine formation, reducing its incidence in the transcribed DNA strand.

Mutational and DNA Binding Specificity of the Carcinogen 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline

The mutagenic specificity of 2-amino-3,8-dimethylimidazo[4, 5-f]quinoxaline (MeIQx), a food-borne mutagen and carcinogen, was studied. Plasmid pK19 was modified by photolysis with the 2-azido form of the carcinogen. High pressure liquid chromatography confirmed that the photoactivated azide formed primarily C8 and N2 guanyl adducts. Transformation of modified pK19 into excision repair competent Escherichia coli resulted in dose-dependent increases in genotoxicity and in mutagenesis within the lacZalpha target sequence. Upon induction of the SOS response, a 20-fold increase in mutation frequency over background was observed. A mutational spectrum for MeIQx, generated by sequencing 125 independent mutants, revealed base substitutions (41%), frameshifts (54%), and complex mutations (5.6%); >90% of the mutations occurred at G-C base pairs. Two hotspots were evident at runs of three or five G-C base pairs; approximately 60% of the mutations occurred at the hotspot sites. The hotspot at position 2532 produced mainly base substitutions, while that at position 2576 gave exclusively frameshift mutations. A polymerase inhibition assay mapped the sites of MeIQx adducts. Arrest sites were primarily at or one base 3' to a guanine residue, which correlated well with the distribution of mutations. No direct correlation was seen, however, between intensity of modification and hotspots for mutation.

Stress concentration factor parametric equations for tubular X and DT joints

During the design stage, the peak stress is usually needed for estimating the fatigue life of offshore tubular welded joints by an S  N approach. However, for fracture mechanics calculations of remaining life, on cracked joints in service, information is required on the magnitude and distribution of the stress acting in the anticipated crack path, not just the peak stress at one location. Fatigue crack propagation rates are important to reliability-based inspection scheduling: hence the need for this information is becoming more pressing. Parametric equations are available for Y and T joints in terms of peak stress, stress distribution and bending-to-membrane ratio. However, for X and DT joints, there are no parametric equations for stress variation through the thickness and around the intersection. Even for stress concentration factor (SCF), so far there is no full set of parametric equations especially for single-brace loading. Thin-shell finite element analyses have been conducted for 330 X and DT joints typical of those used in offshore structures, subject to six modes of loading. The results from this work have been used to produce a new set of parametric equations as a function of non-dimensional joint geometric ratios α, β, γ, τ and θ by carrying out regression analysis. These equations can be used to predict SCFs at the crown toe, saddle, crown heel and hot-spot positions for each mode of loading, for both chord and brace, as well as the angular location of the hot-spot stress site around the intersection. This set of SCF parametric equations has been assessed by comparing the predicted values with results from steel and acrylic model tests and also with the predictions from existing parametric formulae given in the literature. The degree of bending data, and stress distribution data, will be reported in other publications.

Characterization of an apparent hotspot for spontaneous mutation in exon 5 of the Chinese hamster APRT gene

We describe an apparent hotspot for spontaneous deletions and base substitution mutations at a TTC trinucleotide direct repeat/ MboII restriction site in exon 5 of the Chinese hamster APRT gene, in a region with the potential to form a relatively stable, quasipalindromic, stem-loop structure. The recurrent 3 bp TTC deletions observed at this site, which account for approx. 20% of the characterized spontaneous APRT deletions in hemizygous CHO cell lines, represent the only spontaneous deletion events that have been recovered more than once at this locus. A total of 11 independently derived, spontaneous CHO cell APRT mutants with identical 3 bp TTC deletions at this exon 5 MboII site, plus another five mutants that have single base substitutions at this site have been identified among spontaneous mutant collections in several different laboratories. Intriguingly, each of the frequently deleted or mutated bases at this exon 5 deletion hotspot site would correspond to one of the unpaired bases within a single-stranded ‘loop’ region of a stable, quasipalindromic, stem-loop structure that can be formed by intrastrand pairing of inverted repeats in this portion of the APRT gene sequence. An identical TTC trinucleotide direct repeat sequence at the same site in exon 5 of the human APRT gene also appears to be a hotspot for spontaneous deletion.

Single Nucleotide Positions Have Proximal and Distal Influence on UV Mutation Hotspots and Coldspots

Base substitution mutation frequency is influenced by the sequence context surrounding lesions in the DNA. We have been studying ultraviolet mutagenesis in human repair-deficient cells in the supFmarker gene carried in a shuttle vector plasmid. There are prominent hotspots, on opposite strands, at the 5′ TC sites in the eight base palindrome 5′ CTTCGAAG. Recently, we developed a reporter system which permits sequence manipulation in the vicinity of mutational hotspots. We have used the system to characterize the influence of individual positions in the palindrome on the frequency of mutagenesis at the two UV hotspots. In this paper we have determined the contribution of bases at the second and third positions in the palindrome. Changes in bases that were in the primer template duplex when the replication complex encountered the photoproducts at one of the hotspot sites significantly increased or decreased the probability of mutations at the site. We also observed modulation of hotspot activity at other sites as a function of single base changes as much as 80 bases away from the hotspots. In these instances, the site of the changed base was in the unreplicated template ahead of the primer terminus when the polymerase encountered the relevant photoproduct. Our results indicate that sequence context has both proximal and distal consequences for mutagenesis.