Construction of frameshift mutation hot spots within the tetracycline resistance gene of pBR322

The chemical carcinogen, N-2-acetylaminofluorene (AAF) when bound covalently to DNA induces a majority (greater than 90%) of frameshift mutations. The mutations occur with high frequencies at defined sequences (i.e. mutation hot spots). Two classes of mutation hot spots were found: at repetitive sequences and at specific non-repetitive sequences. Mutations at the repetitive sequences depend upon a functional umuC gene whereas mutations at specific non-repetitive sequences are umuC-independent. The first discovered sequence of this class is the NarI restriction enzyme recognition sequence (5'GGCGCC3'). In an attempt to define a family of such sequences we constructed a related sequence 5'GCGCGC3' within the tetracycline resistance gene of pBR322. This sequence was also found to be an--AAF induced--2 frameshift mutation hot spot in both wild type and umuC strains.     

Construction of plasmids containing a unique acetylaminofluorene adduct located within a mutation hot spot. A new probe for frameshift mutagenesis

N-2-acetylaminofluorene (AAF), a potent rat liver carcinogen, binds primarily to the C-8 position of guanine residues. In a bacterial forward mutation assay, more than 90% of the mutations induced by -AAF adducts are frameshift mutations located at specific sites: the so-called mutation hot spots. We are particularly interested in a class of -2 frameshift mutations occurring within a specific sequence, the NarI sequence. The NarI site, GGCGCC, contains three guanine residues that are approximately equally reactive toward -AAF substitution. To study further the mechanism by which mutations are induced by -AAF adducts at this site, we designed a new plasmid probe. In this paper we describe the construction and the effectiveness of this probe, pSM14, which provides a simple phenotypic test for detecting frameshift mutations within the NarI site. The construction and the characterization of plasmids with a single -AAF adduct in each of the three positions of the NarI site are also described. The strategy of construction that was used involves the ligation of oligonucleotides containing a single adduct in a NarI site into a gapped-duplex pSM14 plasmid. Plasmids that have successfully integrated the oligonucleotides by ligation at both the 5' and the 3' ends were purified by centrifugation on CsCl gradients. These constructs have been used in single adduct mutation studies.     

CpG dinucleotides are mutation hot spots in phenylketonuria

The coding region of the phenylalanine hydroxylase (PAH) gene contains 22 CpG dinucleotides, including five doublets in the seventh exon of the gene. We hypothesized that CpG doublets could represent mutation hot spots in PAH deficiencies and we carried out the systematic sequence analysis of exon 7 in 20 unrelated PAH-deficient kindreds of Mediterranean ancestry. This procedure resulted in the detection of two novel missense mutations whose location and nature (CG to CA and CG to TG) were consistent with the accidental deamination of a 5-methylcytosine in a CpG doublet (codon 261arg----gln and codon 252arg----trp). Moreover, the codon 261 mutation was found to be associated with mutant restriction fragment length polymorphism (RFLP) haplotype 1, the most frequent mutant RFLP haplotype at the PAH locus in the studies reported thus far. However, since the mutation was detected in only 36% of haplotype 1 mutant alleles, it appears that this haplotype at the PAH locus is genotypically heterogeneous in Mediterranean countries.     

A umuDC-independent SOS pathway for frameshift mutagenesis.

Summary The chemical carcinogen N-acetoxy-N-2-acetylaminofluorene induces mainly frameshift mutations, which occur within two types of sequences (mutation hot spots): –1 frameshift mutations within contiguous guanine sequences and –2 frameshift mutations within alternating GC sequences such as the NarI and BssHII restriction site sequences. We have investigated the genetic control of mutagenesis at these sequences by means of a reversion assay using plasmids pW17 and pX2, which contain specific targets for contiguous guanine and alternating GC sequences, respectively. Our results suggest that mutations at these hot spot sequences are generated by two different genetic pathways, both involving induction of SOS functions. The two pathways differ both in their LexA-controlled gene and RecA protein requirements. In the mutation pathway that acts at contiguous guanine sequences, the RecA protein participates together with the umuDC gene products. In contrast, RecA is not essential for mutagenesis at alternating GC sequences, except to cleave the LexA repressor. The LexA-regulated gene product(s), which participate in this latter mutational pathway, do not involve umuDC but another as yet uncharacterized inducible function. We also show that wild-type RecA and RecA430 proteins exert an antagonistic effect on mutagenesis at alternating GC sequences, which is not observed either in the presence of activated RecA (RecA^*), RecA730 or RecA495 proteins, or in the complete absence of RecA as in recA99. It is concluded that the –1 mutation pathway presents the same genetic requirements as the pathway for UV light mutagenesis, while the –2 mutation pathway defines a distinct SOS pathway for frameshift mutagenesis.     

Spontaneous and 9-aminoacridine-induced frameshift mutagenesis: second-site frameshift mutation within the N-terminal region of the lacI gene of Escherichia coli

Summary A novel forward mutational system, based on the acquisition of an I^q-d dominant phenotype from an initial I^q− recessive state, was used to identify second-site frameshift mutation [±1(±3 _n ) events] within the N-terminal region of thelacI gene ofEscherichia coli. The DNA sequences are described of forty-six spontaneous and twenty 9-aminoacridine(9-AA)-induced second site mutations. Although −1 frameshift events dominate both spectra, the nature and site specificity of these events clearly distinguish two mutational distributions. The spontaneous distribution contains two −(A: T) frameshift hotspots; one within a monotonic A₅ run (9 occurrences), the other at a 5′-CACAACAAC-3′ sequence (12 occurrences). In contrast 17 of the 20 mutations recovered after 9-AA treatment involve the loss of a G: C pair, 14 of which occur at a single site (5′-CGGGC-3′). The striking specificity of the observed mutational hotspots is of interest since this open genetic target contains similar sequences which were infrequently recovered.     

Conservation of polar residues as hot spots at protein interfaces

A number of studies have addressed the question of which are the critical residues at protein-binding sites. These studies examined either a single or a few protein-protein interfaces. The most extensive study to date has been an analysis of alanine-scanning mutagenesis. However, although the total number of mutations was large, the number of protein interfaces was small, with some of the interfaces closely related. Here we show that although overall binding sites are hydrophobic, they are studded with specific, conserved polar residues at specific locations, possibly serving as energy "hot spots." Our results confirm and generalize the alanine-scanning data analysis, despite its limited size. Previously Trp, Arg, and Tyr were shown to constitute energetic hot spots. These were rationalized by their polar interactions and by their surrounding rings of hydrophobic residues. However, there was no compelling reason as to why specifically these residues were conserved. Here we show that other polar residues are similarly conserved. These conserved residues have been detected consistently in all interface families that we have examined. Our results are based on an extensive examination of residues which are in contact across protein interfaces. We utilize all clustered interface families with at least five members and with sequence similarity between the members in the range of 20-90%. There are 11 such clustered interface families, comprising a total of 97 crystal structures. Our three-dimensional superpositioning analysis of the occurrences of matched residues in each of the families identifies conserved residues at spatially similar environments. Additionally, in enzyme inhibitors, we observe that residues are more conserved at the interfaces than at other locations. On the other hand, antibody-protein interfaces have similar surface conservation as compared to their corresponding linear sequence alignment, consistent with the suggestion that evolution has optimized protein interfaces for function.     

Inactivation of nonsense suppressor transfer RNA genes in Schizosaccharomyces pombe. Intergenic conversion and hot spots of mutation

Intergenic conversion is a mechanism for the concerted evolution of repeated DNA sequences. A new approach for the isolation of intergenic convertants of serine tRNA genes in the yeast Schizosaccharomyces pombe is described. Contrary to a previous scheme, the intergenic conversion events studied in this case need not result in functional tRNA genes. The procedure utilizes crosses of strains that are homozygous for an active UGA suppressor tRNA gene, and the resulting progeny spores are screened for loss of suppressor activity. In this way, intergenic convertants of a tRNA gene are identified that inherit varying stretches of DNA sequence from either of two other tRNA genes. The information transferred between genes includes anticodon and intron sequences. Two of the three tRNA genes involved in these information transfers are located on different chromosomes. The results indicate that intergenic conversion is a conservative process. No infidelity is observed in the nucleotide sequence transfers. This provides further evidence for the hypothesis that intergenic conversion and allelic conversion are the result of the same molecular mechanism. The screening procedure for intergenic revertants also yields spontaneous mutations that inactivate the suppressor tRNA gene. Point mutations and insertions of A occur at various sites at low frequency. In contrast, A insertions at one specific site occur with high frequency in each of the three tRNA genes. This new type of mutation hot spot is found also in vegetative cells.     

Ethidium bromide enhancement of frameshift mutagenesis caused by photoactivatable ethidium analogs.

Ethidium azide analogs (3-amino-8-azido-ethidium monoazide and ethidium diazide) have been developed as photosensitive probes in order to analyze directly the reversible in vivo interactions of ethidium bromide. Our preliminary observations [11], relating the mutagenic potential of the monoazide analog of ethidium, have been extended and refined, using the highly purified ethidium azide analogs [5]. A number of physical-chemical studies indicate that the monoazide analog interaction with nucleic acids, prior to photolysis, resembles remarkably the interaction of the parent ethidium (unpublished). It was anticipated, therefore, that competition by ethidium for the ethidium monoazide mutagenic sites in Salmonella TA1538 would be observed when these drugs were used in combination. Previous results in fact showed a decreased production of frameshift mutants when ethidium bromide was added to the ethidium monoazide in the Ames assay [1]. However, more extensive investigations, reported here, have shown that this apparent competition was the result of neglecting the toxic effects of ethidium monoazide and its enhanced toxocity in the presence of ethidium bromide. Conversely, an enhancement of the azide mutagenesis and toxicity for both the mono- and diazide analogs was seen when ethidium bromide was used in combination with these analogs.     

Increased thermostability of microbial transglutaminase by combination of several hot spots evolved by random and saturation mutagenesis

The thermostability of microbial transglutaminase (MTG) of Streptomyces mobaraensis was further improved by saturation mutagenesis and DNA-shuffling. High-throughput screening was used to identify clones with increased thermostability at 55°C. Saturation mutagenesis was performed at seven "hot spots", previously evolved by random mutagenesis. Mutations at four positions (2, 23, 269, and 294) led to higher thermostability. The variants with single amino acid exchanges comprising the highest thermostabilities were combined by DNA-shuffling. A library of 1,500 clones was screened and variants showing the highest ratio of activities after incubation for 30 min at 55°C relative to a control at 37°C were selected. 116 mutants of this library showed an increased thermostability and 2 clones per deep well plate were sequenced (35 clones). 13 clones showed only the desired sites without additional point mutations and eight variants were purified and characterized. The most thermostable mutant (triple mutant S23V-Y24N-K294L) exhibited a 12-fold higher half-life at 60°C and a 10-fold higher half-life at 50°C compared to the unmodified recombinant wild-type enzyme. From the characterization of different triple mutants differing only in one amino acid residue, it can be concluded that position 294 is especially important for thermostabilization. The simultaneous exchange of amino acids at sites 23, 24, 269 and 289 resulted in a MTG-variant with nearly twofold higher specific activity and a temperature optimum of 55°C. A triple mutant with amino acid substitutions at sites 2, 289 and 294 exhibits a temperature optimum of 60°C, which is 10°C higher than that of the wild-type enzyme.     

Deletion hot spots in chimeric Escherichia coli plasmids.

Deletions form frequently in chimeric plasmids composed of M13mp2, pBR322, and pC194 (B. Michel and S. D. Ehrlich, Proc. Natl. Acad. Sci. USA 83:3386-3390, 1986). They are generated by joining of the nucleotide neighboring the nick site in the M13 replication origin to a nonadjacent nucleotide. This nucleotide is most often located within particular short plasmid regions, named deletion hot spots. Three natural hot spots were present in the chimeric plasmids. Two were active only when the DNA replication initiated at the M13 origin was allowed to progress; the third was active only in the presence of wild-type amounts of DNA ligase. Three artificial hot spots were generated by creating palindromic sequences in the plasmids.     

Hereditary desmoid disease due to a frameshift mutation at codon 1924 of the APC gene.

Desmoid tumors are slowly growing fibrous tumors highly resistant to therapy and often fatal. Here, we report hereditary desmoid disease (HDD), a novel autosomal dominant trait with 100% penetrance affecting a three-generation kindred. Desmoid tumors are usually a complication of familial adenomatous polyposis, a predisposition to the early development of premalignant adenomatous polyps in the colorectum due to chain-terminating mutations of the APC gene. In general, one or more members in approximately 10% of the FAP families manifest desmoid tumors. Affected individuals from the HDD kindred are characterized by multifocal fibromatosis of the paraspinal muscles, breast, occiput, arms, lower ribs, abdominal wall, and mesentery. Osteomas, epidermal cysts, and other congenital features were also observed. We show that HDD segregates with an unusual germ-line chain-terminating mutation at the 3' end of the APC gene (codon 1924) with somatic loss of the wild-type allele leading to tumor development.     

Distinct functions of MLH3 at recombination hot spots in the mouse

The four mammalian MutL homologs (MLH1, MLH3, PMS1, and PMS2) participate in a variety of events, including postreplicative DNA repair, prevention of homeologous recombination, and crossover formation during meiosis. In this latter role, MLH1-MLH3 heterodimers predominate and are essential for prophase I progression. Previous studies demonstrated that mice lacking Mlh1 exhibit a 90% reduction in crossing over at the Psmb9 hot spot while noncrossovers, which do not result in exchange of flanking markers but arise from the same double-strand break event, are unaffected. Using a PCR-based strategy that allows for detailed analysis of crossovers and noncrossovers, we show here that Mlh3(-/-) exhibit a 85-94% reduction in the number of crossovers at the Psmb9 hot spot. Most of the remaining crossovers in Mlh3(-/-) meiocytes represent simple exchanges similar to those seen in wild-type mice, with a small fraction (6%) representing complex events that can extend far from the initiation zone. Interestingly, we detect an increase of noncrossovers in Mlh3(-/-) spermatocytes. These results suggest that MLH3 functions predominantly with MLH1 to promote crossovers, while noncrossover events do not require these activities. Furthermore, these results indicate that approximately 10% of crossovers in the mouse are independent of MLH3, suggesting the existence of alternative crossover pathways in mammals.     

Stabilization of the intermediate in frameshift mutation

A mismatch repair, proofreading deficient mutant of Escherichia coli lost a C from a C8 run at a rate 10 times higher than the loss of A from an A8 sequence in the same double mutant. This greater frameshift instability of a homopolymeric run of C's may be due to stabilization of a stacked intermediate. Gain of a (CA) unit in a similarly constructed (CA)15 sequence occurred at a rate about 1/3 that previously reported for a (CA)14 construct losing a (CA) repeat unit.     

Increased recombination intermediates and homologous integration hot spots at DNA replication origins

We have studied the relationship between DNA replication and recombination in Schizosaccharomyces pombe using two-dimensional gel electrophoresis and functional analysis. Our results indicate that the activation of replication origins (ORIs) during the mitotic cell cycle is associated with the generation of joint DNA molecules between sister chromatids. The frequency of integration by homologous recombination was up to 50-fold higher than the genomic average within a narrow window overlapping the ars1 replication initiation site. The S. pombe rad22Delta, rhp51Delta, and rhp54Delta mutants, deficient in mitotic recombination, activate ORIs very inefficiently and accumulate abnormal replication intermediates. These results focus on the general link between replication and recombination previously found in several systems and suggest a role for recombination in the initiation of eukaryotic DNA replication.     

A structural model for sequence-specific proflavin-DNA interactions during in vitro frameshift mutagenesis.

Molecular models describing intermediates that may lead to proflavin-induced 1 bp deletions during in vitro polymerization by E. coli DNA polymerase I Klenow fragment are proposed. The models provide structural explanations for the fact that the induced frameshifts always occur opposite template bases that are adjacent to 5' pyrimidines and are based on the underlying hypothesis that the deletions arise because the polymerase passes by a template base without copying it. Because the most frequent mutations are opposite Pu in the template sequence 5' Py Pu 3', a single-strand loop-out model was constructed for this sequence and proflavin was added, using structures found in crystalline oligonucleotides and their complexes with proflavin. The model seeks to rationalize the roles of the 5' pyrimidine and proflavin in facilitating the bypass. Four potential roles for proflavin in mutagenesis are described: 1) stacking on the looped-out base; 2) stacking on the base pair immediately preceding the site of mutation; 3) hydrogen bonding with the 5' pyrimidine; 4) hydrogen bonding with the phosphate backbone. These models point to the possibility that a number of proflavin-DNA interactions may be involved. In contrast, modeling does not suggest a role for classically intercalated proflavin in frameshift mutagenesis arising during in vitro DNA polymerization.     

Constitutively methylated CpG dinucleotides as mutation hot spots in the retinoblastoma gene (RB1).

A wide spectrum of mutations, ranging from point mutations to large deletions, have been described in the retinoblastoma gene (RB1). Mutations have been found throughout the gene; however, these genetic alterations do not appear to be homogeneously distributed. In particular, a significant proportion of disease-causing mutations results in the premature termination of protein synthesis, and the majority of these mutations occur as C-->T transitions at CpG dinucleotides (CpGs). Such recurrent CpG mutations, including those found in RB1, are likely the result of the deamination of 5-methylcytosine within these CpGs. In the present study, we used the sodiumbisulfite conversion method to detect cytosine methylation in representative exons of RB1. We analyzed DNA from a variety of tissues and specifically targeted CGA codons in RB1, where recurrent premature termination mutations have been reported. We found that DNA methylation within RB1 exons 8, 14, 25, and 27 appeared to be restricted to CpGs, including six CGA codons. Other codons containing methylated cytosines have not been reported to be mutated. Therefore, disease-causing mutations at CpGs in RB1 appear to be determined by several factors, including the constitutive presence of DNA methylation at cytosines within CpGs, the specific codon within which the methylated cytosine is located, and the particular region of the gene within which that codon resides.