Double-strand breaks increase the incidence of genetic deletion associated with intermolecular recombination in bacteriophage T7

An in vitro DNA replication system based on extracts prepared from Escherichia coli cells infected with bacteriophage T7 was used to study deletion associated with the repair of double-strand breaks. The gene for T7 ligase was interrupted by a DNA insert which included 17-bp direct repeats. Deletion between the repeats restored the reading frame of the gene, and these DNA molecules could be detected by their ability to give rise to ligase-positive phage after in vitro packaging. T7 genomes that had a pre-existing double-strand break located between the direct repeats were incubated together with intact genomes which had the same direct repeats. Genetic markers placed on either side of the insert in the ligase gene allowed identification of the source of DNA molecules that underwent deletion between the direct repeats. This allowed an assessment of the participation of the molecules with strand breaks in the deletion process, under conditions where any mechanism could contribute to deletion. Approximately three-quarters of the T7 molecules that had lost the region between the direct repeats contained one or both of the partial genomes originally introduced into the reactions. About 50% of the genomes which had undergone deletion had recombined markers between the partial and intact genomes. The data demonstrate that double-strand breaks substantially enhance the contribution of intermolecular recombination to deletion. Received: 19 November 1996 / Accepted: 26 February 1997     

The Influence of Primary and Secondary DNA Structure in Deletion and Duplication between Direct Repeats in Escherichia Coli

We describe a system to measure the frequency of both deletions and duplications between direct repeats. Short 17- and 18-bp palindromic and nonpalindromic DNA sequences were cloned into the EcoRI site within the chloramphenicol acetyltransferase gene of plasmids pBR325 and pJT7. This creates an insert between direct repeated EcoRI sites and results in a chloramphenicol-sensitive phenotype. Selection for chloramphenicol resistance was utilized to select chloramphenicol resistant revertants that included those with precise deletion of the insert from plasmid pBR325 and duplication of the insert in plasmid pJT7. The frequency of deletion or duplication varied more than 500-fold depending on the sequence of the short sequence inserted into the EcoRI site. For the nonpalindromic inserts, multiple internal direct repeats and the length of the direct repeats appear to influence the frequency of deletion. Certain palindromic DNA sequences with the potential to form DNA hairpin structures that might stabilize the misalignment of direct repeats had a high frequency of deletion. Other DNA sequences with the potential to form structures that might destabilize misalignment of direct repeats had a very low frequency of deletion. Duplication mutations occurred at the highest frequency when the DNA between the direct repeats contained no direct or inverted repeats. The presence of inverted repeats dramatically reduced the frequency of duplications. The results support the slippage-misalignment model, suggesting that misalignment occurring during DNA replication leads to deletion and duplication mutations. The results also support the idea that the formation of DNA secondary structures during DNA replication can facilitate and direct specific mutagenic events.     

Deletion between direct repeats in T7 DNA stimulated by double-strand breaks.

An in vitro system based on extracts of Escherichia coli infected with bacteriophage T7 was used to study genetic deletions between directly repeated sequences. The frequency of deletion was highest under conditions in which the DNA was actively replicating. Deletion frequency increased markedly with the length of the direct repeat both in vitro and in vivo. When a T7 gene was interrupted by 93 bp of nonsense sequence flanked by 20-bp direct repeats, the region between the repeats was deleted in about 1 out of every 1,600 genomes during each round of replication. Very similar values were found for deletion frequency in vivo and in vitro. The deletion frequency was essentially unaffected by a recA mutation in the host. When a double-strand break was placed between the repeats, repair of this strand break was often accompanied by the deletion of the DNA between the direct repeats, suggesting that break rejoining could contribute to deletion during in vitro DNA replication.     

Palindromy and the Location of Deletion Endpoints in Escherichia Coli

The contributions of direct and inverted repeats to deletion formation were studied by characterizing Ampr revertants of plasmids with a series of insertion mutations at a specific site in the pBR322 ampicillin resistance (amp) gene. The inserts at this site are palindromic, variable in length, and bracketed by 9- or 10-bp direct repeats of amp sequence. There is an additional direct repeat composed of 4 bp within the insert and 4 bp of adjoining amp sequence. DNA sequencing and colony hybridization of Ampr revertants showed that they contained either the parental amp sequence, implying deletion endpoints in the flanking 9- or 10-bp repeats, or a specific 1-bp substitution, implying endpoints in the 4-bp repeats. Although generally direct repeats seem to be used as deletion endpoints with a frequency proportional to their lengths, we found that with uninterrupted palindromes longer than 32 bp, the majority of deletions ended in the 4 bp, not the 9- or 10-bp repeats. This preferential use of the shorter direct repeats associated with palindromes is interpreted according to a DNA synthesis-error model in which hairpin structures formed by intrastrand pairing foster the slippage of nascent strands during DNA synthesis.     

Detection of a specific mitochondrial DNA deletion in tissues of older humans.

Using PCR, we found that normal heart muscle and brain from adult human individuals contain low levels of a specific mitochondrial DNA deletion, previously found only in patients affected with certain types of neuromuscular disease. This deletion was not observed in fetal heart or brain. Experimental tests support the idea that the deletion exists in vivo in adult mitochondria and is not an in vitro artifact of PCR. Our data provide direct experimental support for the idea that accumulation of mitochondrial DNA deletions may be important in aging.     

WT1 exon 1 deletion/insertion mutations in Wilms tumor patients, associated with di- and trinucleotide repeats and deletion hotspot consensus sequences.

The WT1 gene is known to play a role in at least some cases of Wilms tumor (WT). The first exon of the gene is highly GC rich and contains many short tandem di- and trinucleotide repeats, interrupted direct repeats, and CCTG (CAGG) motifs that have been identified as hotspots for DNA deletions. We have analyzed 80 WT patient samples for mutations in the first exon of WT1, either by SSCP analysis of the first 131 bp of the coding portion of WT1 exon 1 or by size analysis of a PCR product encompassing the coding region of exon 1 in addition to flanking noncoding regions. We report here the occurrence of somatic and germ-line deletion and insertion mutations in this portion of the gene in four WT patients. The mutations are flanked by short direct repeats, and the breakpoints are within 5 nt of a CCTG (CAGG) sequence. These data suggest that a distinctive mutational mechanism, previously unrecognized for this gene, is important for the generation of DNA mutations at the WT1 locus.     

Identification of a nonframeshift 84-bp deletion in exon 13 of the cystic fibrosis gene.

Cystic fibrosis (CF) is the most frequent autosomal recessive inherited disorder in Caucasian populations. The disease is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have identified an 84-bp deletion in exon 13 of the CFTR gene, detected by DNA amplification and direct sequencing of 500 bp of the 5' end of exon 13. The deletion was in the maternal allele of a CF patient bearing the delta F508 deletion in the father's allele. The same 84-bp deletion could also be detected in the patient's mother. The deletion spanned from a four-A cluster in positions 1949-1952 to another four-A cluster in positions 2032-2035, including 84 bp which correspond to codons 607-634 (1949del84). The reported mutation would result in the loss of 28 amino acid residues of the R domain of the CFTR protein.     

Deletion formation in bacteriophage T4

We have manipulated the dispensable region of the rIIB gene of bacteriophage T4 in order to study the generation of deletions involving direct repeats. We show that recombination between different parental chromosomes is one source of the deletions we have studied. We have also investigated the effects of structure, base composition and distance on deletion formation. We demonstrate that the potential to form structure in single-stranded DNA has variable effects on the frequency of deletion formation and conclude that, in some cases, slipped mispairing during DNA synthesis can make a substantial contribution to deletion frequencies. The G + C richness of the direct repeats involved in deletion formation is an important parameter of the frequency of deletion formation. We have confirmed that increasing the distance between direct repeats decreases deletion frequency.     

High-frequency spontaneous deletion of DNA sequences flanked by direct DNA repeats which also contain an internal palindrome

The DNA sequences associated with a very high-frequency, spontaneous deletion event have been determined to be two 11-base direct repeats which also contain an internal 6-base palindrome. A parental M13 replicative form (RF) DNA harboring DNA fragments of the T4 denV gene contained these direct repeats and could only be maintained at 5% of the total RF DNA within an infected cell. The remaining RF DNA was deleted for all intervening sequences between the direct repeats (2.2-kb), but one copy of the direct repeat was retained after the deletion had occurred. This site-specific deletion was highly reproducible in that if parental-sized M13 RF DNA was gel purified and transformed back into cells, the deletion occurred at precisely the same sequence as before. Electron microscopic analyses of DNA extracted from cells transformed with parental-sized DNA revealed the presence of excised 2.2-kb double-stranded circular DNA molecules. This observation thus rules out a copy choice replication/deletion mechanism to account for this high-frequency deletion event.     

Gene deletions causing human genetic disease: mechanisms of mutagenesis and the role of the local DNA sequence environment

Summary Reports describing short (< 20 bp) gene deletions causing human genetic disease were collated in order to study underlying causative mechanisms. Deletion break-point junction regions were found to be non-random both at the nucleotide and dinucleotide sequence levels, an observation consistent with an endogenous sequencedirected mechanism of mutagenesis. Direct repeats of between 2 bp and 8 bp were found in the immediate vicinity of all but one of the 60 deletions analysed. Direct repeats are a feature of a number of recombination, replication or repair-based models of deletion mutagenesis and the possible contribution of each to the spectrum of mutations examined was assessed. The influence of parameters such as repeat length and lenght of DNA between repeats was studied in relation to the frequency, location and extent of these deletions. Findings were broadly consistent with a slipped mispairing model but the predicted deletion of one whole repeat copy was found only rarely. A modified version of the slipped mispairing hypothesis was therefore proposed and was shown to possess considerable explanatory value for 25% of deletions examined. Whereas the frequency of inverted repeats in the vicinity of gene deletions was not significantly elevated, these elements may nevertheless promote instability by facilitating the formation of secondary structure intermediates. A significant excess of symmetrical sequence elements was however found at sites of single base deletions. A new model to explain the involvement of symmetric elements in frameshift mutagenesis was devised, which successfully accounted for a majority of the single base deletions examined. In general, the loss of one or a few base pairs of DNA was found to be more compatible with a replication-based model of mutagenesis than with a recombination or repair hypothesis. Seven hitherto unrecognized hotspots for deletion were noted in five genes (AT3, F8, HBA, HBB and HPRT). Considerable sequence homology was found between these different sites, and a consensus sequence (TGA/GA/GG/ TA/C) was drawn up. Sequences fitting this consensus (i) were noted in the immediate vicinity of 41% of the other (sporadic) gene deletions, (ii) were found frequently at sites of spontaneous deletion in the hamster APRT gene, (iii) were found to be associated with many larger human gene deletions/translocations, (iv) act as arrest sites for human polymerase a during DNA replication and (v) have been shown by in vitro studies of human polymerase a to be especially prone to frameshift mutation. It is proposed that dissociation of polymerase a at arrest sites may, by providing a stable single stranded substrate, lead to deletion of a DNA sequence either by slipped mispairing via a number of different secondary structure intermediates, or by strand-switching or base misincorporation. Human gene deletions thus appear to be caused by multiple mechanisms whose relative importance is probably governed by local primary and secondary DNA structure. Our ability to predict precisely the location and extent of a gene deletion is however hampered both by this complexity and by the possibility that these mechanisms may often act in combination.     

Molecular analyses of mtDNA deletion mutations in microdissected skeletal muscle fibers from aged rhesus monkeys

Mitochondrial DNA (mtDNA) deletion mutations co-localize with electron transport system (ETS) abnormalities in rhesus monkey skeletal muscle fibers. Using laser capture microdissection in conjunction with PCR and DNA sequence analysis, mitochondrial genomes from single sections of ETS abnormal fibers were characterized. All ETS abnormal fibers contained mtDNA deletion mutations. Deletions were large, removing 20-78% of the genome, with some to nearly all of the functional genes lost. In one-third of the deleted genomes, the light strand origin was deleted, whereas the heavy strand origin of replication was conserved in all fibers. A majority (27/39) of the deletion mutations had direct repeat sequences at their breakpoints and most (36/39) had one breakpoint within or in close proximity to the cytochrome b gene. Several pieces of evidence support the clonality of the mtDNA deletion mutation within an ETS abnormal region of a fiber: (a) only single, smaller than wild-type, PCR products were obtained from each ETS abnormal region; (b) the amplification of mtDNA from two regions of the same ETS abnormal fiber identified identical deletion mutations, and (c) a polymorphism was observed at nucleotide position 16103 (A and G) in the wild-type mtDNA of one animal (sequence analysis of an ETS abnormal region revealed that mtDNA deletion mutations contained only A or G at this position). Species-specific differences in the regions of the genomes lost as well as the presence of direct repeat sequences at the breakpoints suggest mechanistic differences in deletion mutation formation between rodents and primates.     

Evidence for two mechanisms of palindrome-stimulated deletion in Escherichia coli: single-strand annealing and replication slipped mispairing

Spontaneous deletion mutations often occur at short direct repeats that flank inverted repeat sequences. Inverted repeats may initiate genetic rearrangements by formation of hairpin secondary structures that block DNA polymerases or are processed by structure-specific endonucleases. We have investigated the ability of inverted repeat sequences to stimulate deletion of flanking direct repeats in Escherichia coli. Propensity for cruciform extrusion in duplex DNA correlated with stimulation of flanking deletion, which was partially sbcD dependent. We propose two mechanisms for palindrome-stimulated deletion, SbcCD dependent and SbcCD independent. The SbcCD-dependent mechanism is initiated by SbcCD cleavage of cruciforms in duplex DNA followed by RecA-independent single-strand annealing at the flanking direct repeats, generating a deletion. Analysis of deletion endpoints is consistent with this model. We propose that the SbcCD-independent pathway involves replication slipped mispairing, evoked from stalling at hairpin structures formed on the single-stranded lagging-strand template. The skew of SbcCD-independent deletion endpoints with respect to the direction of replication supports this hypothesis. Surprisingly, even in the absence of palindromes, SbcD affected the location of deletion endpoints, suggesting that SbcCD-mediated strand processing may also accompany deletion unassociated with secondary structures.     

Mechanisms of deletion formation in Escherichia coli plasmids. II. Deletions mediated by short direct repeats.

Summary A set of plasmids containing 42, 21 and 13 bp direct repeats was used to analyze the effect of repeat length on the frequencies of deletion formation and the structure of the deleted derivatives of different recombination-deficient Escherichia coli strains. Agarose gel electrophoresis of plasmid DNA demonstrated that the formation of deletions in these plasmids was associated with dimerization of plasmid DNA. Restriction analysis of the dimers showed that deletions at short direct repeats arose non-conservatively, that is, the formation of a deletion in one monomeric plasmid unit was not associated with a duplication in the other. Mutations in the recA, recF, recJ and recO genes had no marked effect on either the frequencies of deletion formation or the structure of dimers. In contrast, recB recC mutations greatly increased the frequencies of deletion formation, 6-fold for 42 bp, and 115-fold for 21 by direct repeats. Conversion of DNA replication to the rolling circle mode in a recB recC strain, resulting in the formation of double-stranded ends, is suggested as the stimulatory effector.     

Replication slippage between distant short repeats in Saccharomyces cerevisiae depends on the direction of replication and the RAD50 and RAD52 genes.

Small direct repeats, which are frequent in all genomes, are a potential source of genome instability. To study the occurrence and genetic control of repeat-associated deletions, we developed a system in the yeast Saccharomyces cerevisiae that was based on small direct repeats separated by either random sequences or inverted repeats. Deletions were examined in the LYS2 gene, using a set of 31- to 156-bp inserts that included inserts with no apparent potential for secondary structure as well as two quasipalindromes. All inserts were flanked by 6- to 9-bp direct repeats of LYS2 sequence, providing an opportunity for Lys+ reversion via precise excision. Reversions could arise by extended deletions involving either direct repeats or random sequences and by -1-or +2-bp frameshift mutations. The deletion breakpoints were always associated with short (3- to 9-bp) perfect or imperfect direct repeats. Compared with the POL+ strain, deletions between small direct repeats were increased as much as 100-fold, and the spectrum was changed in a temperature-sensitive DNA polymerase delta pol3-t mutant, suggesting a role for replication. The type of deletion depended on orientation relative to the origin of replication. On the basis of these results, we propose (i) that extended deletions between small repeats arise by replication slippage and (ii) that the deletions occur primarily in either the leading or lagging strand. The RAD50 and RAD52 genes, which are required for the recombinational repair of many kinds of DNA double-strand breaks, appeared to be required also for the production of up to 90% of the deletions arising between separated repeats in the pol3-t mutant, suggesting a newly identified role for these genes in genome stability and possibly replication.     

A Source of Small Repeats in Genomic DNA

The processes of spontaneous mutation are known to be influenced by neighboring DNA. Imperfect nearby repeats in the neighboring DNA have been observed to mutate to form perfect repeats. The repeats may be either direct or inverted. Such a mutational process should create perfect direct and inverted repeats in intergenic DNA. A larger than expected number of direct repeats has generally been observed in a wide range of species in both coding and noncoding DNA. Simulations are carried out to determine how this process might influence the repetitive structure of genomic DNA. These simulations show that small repeats created by this kind of a mutational process can explain the excess number of repeats in intergenic DNA. The simulations suggest that this mechanism may be a common cause of mutations, including single-base changes. The influences of the distance between imperfect repeats and of their degree of similarity are investigated.     

Comparison of the waxy locus sequence from a non-waxy strain and two waxy mutants of spontaneous and artificial origins in barley

Molecular characterization of 3 alleles of the waxy gene from a non-waxy strain "Shikoku hadaka No. 84" (SH84), an indigenous waxy strain "Mochimugi D" (MMD), and an artificial waxy mutant strain "Shikoku hadaka No. 97" (SH97) of barley (Hordeum vulgare ssp. vulgare) was performed via a PCR direct sequencing strategy. The 3 haplotypes were analyzed in terms of single nucleotide polymorphisms, insertion/deletion mutations, and simple sequence repeat polymorphisms. In comparison with the barley non-waxy gene sequence deposited in the public DNA database, 110 polymorphic sites were found in the 5,190-bp sequenced region of the non-waxy strain SH84. A 418-bp deletion in the 5' non-coding sequence was identified in the indigenous waxy strain MMD. Except for the deletion in the promoter region, the spontaneous mutant wax allele and non-waxy allele were identical. Such highly conserved sequences provide evidence for the recent occurrence of a deletion event in the cultivated barley gene pool. Compared to the original variety SH84, induced waxy mutant SH97 had a base substitution of a C to T in the exon 5, which converting Gln-89 of the wild-type gene into a stop codon, suggesting the involvement of a nonsense-mediated mRNA decay. These results will be helpful for understanding the mechanism of the variable amylose content in waxy cultivars of cereal species.     

The effect of the 3′ → 5′ exonuclease of T7 DNA polymerase on frameshifts and deletions

Both spontaneous frameshift mutation and deletion mutation were measured in a T7 phage deficient in the 3′ → 5′ exonuclease of T7 DNA polymerase. It was found that the absence of this exonuclease caused a marked increase in the revision of both plus one and minus one mutations. The exonuclease deficiency caused essentially no effect on the frequency of deletion between 10-bp direct repeats even when the segment between the direct repeats contained a 25-bp palindrome.     

A fragile X case with an amplification/deletion mosaic pattern

Fragile X syndrome is the most common cause of hereditary mental retardation. The FMR1 gene, which is involved in fragile X syndrome, contains a polymorphic CGG repeat, which expands in affected patients. Expanding triplet repeats have been shown to be a new type of mutation, termed "dynamic mutation", responsible for more than 12 genetic diseases. These mutations occur as multiple steps rather than as a single event. The first step leads to an unstable allele that then becomes increasingly unstable generally achieving further increases in copy or occasionally contraction. In this report, we describe a fragile X boy with both a hypermethylated full mutation and a deletion of 905 bp encompassing the CGG repeat. The upstream breakpoint is 438 bp 5' to the CGG repeat and the downstream breakpoint is 420 bp 3' of the triplet repeats. The deletion includes the ATG starting codon for translation of the FMR1 gene. This was confirmed by using FMRP immunocytochemistry both on blood smears and hair roots. The deleted region is flanked by a ccgg direct repeat next to the breakpoints; this may have had a critical role in the formation of a secondary DNA structure leading to the deletion.     

Identical 3250-bp deletion between two AluI repeats in the ADA genes of unrelated ADA-SCID patients.

Recently, we investigated a Belgian patient with severe combined immune deficiency caused by a dysfunction of the gene for adenosine deaminase (ADA-SCID), which was found to be due to a 3.2-kb deletion spanning the promoter and the first exon of the ADA gene (Berkvens et al., 1987, Eur. J. Pediatr. 146:329). No ADA-specific RNA could be detected in primary fibroblasts derived from this patient. In the present paper we establish via direct sequencing of in vitro amplified DNA that the 3250-bp deletion is due to a recombination within the left arms of two direct AluI repeats. This mutation is identical to one reported for an unrelated patient in the United States (Markert et al., 1988, J. Clin. Invest. 81:1323-1327).