Mutations in Pol1 Increase the Mitotic Instability of Tandem Inverted Repeats in Saccharomyces Cerevisiae

Tandem inverted repeats (TIRs or hairpins) of 30 and 80 base-pair unit lengths are unstable mitotically in yeast (Saccharomyces cerevisiae). TIR instability results from deletions that remove part or all of the presumed hairpin structure from the chromosome. At least one deletion endpoint is always at or near the base of the hairpin, and almost all of the repaired junctions occur within short direct sequence repeats of 4 to 9 base pairs. The frequency of this event, which we call ``hairpin excision,' is influenced by chromosomal position, length of the inverted repeats, and the distance separating the repeat units; increasing the distance between the inverted repeats as little as 25 base pairs increases their chromosomal stability. The frequency of excision is not affected by representative rad mutations, but is influenced by mutations in certain genes affecting DNA synthesis. In particular, mutations in POL1/CDC17, the gene that encodes the large subunit of DNA polymerase I, increase the frequency of hairpin deletions significantly, implicating this protein in the normal maintainance of genomic TIRs.     

Role of Reciprocal Exchange, One-Ended Invasion Crossover and Single-Strand Annealing on Inverted and Direct Repeat Recombination in Yeast: Different Requirements for the Rad1, Rad10, and Rad52 Genes

We have constructed novel DNA substrates (one inverted and three direct repeats) based on the same 0.6-kb repeat sequence to study deletions and inversions in Saccharomyces cerevisiae. Spontaneous deletions occur six to eight times more frequently than inversions, irrespective of the distance between the repeats. This difference can be explained by the observation that deletion events can be mediated by a recombination mechanism that can initiate within the intervening sequence of the repeats. Spontaneous and double-strand break (DSB) -induced deletions occur as RAD52-dependent and RAD52-independent events. Those deletion events initiated through a DSB in the unique intervening sequence require the Rad1/Rad10 endonuclease only if the break is distantly located from the flanking DNA repeats. We propose that deletions can occur as three types of recombination events: the conservative RAD52-dependent reciprocal exchange and the nonconservative events, one-ended invasion crossover, and single-strand annealing (SSA). We suggest that one-ended invasion is RAD52 dependent, whereas SSA is RAD52 independent. Whereas deletions, like inversions, occur through reciprocal exchange, deletions can also occur through SSA or one-ended invasion. We propose that the contribution of reciprocal exchange and one-ended invasion crossover vs. SSA events to overall spontaneous deletions is a feature specific for each repeat system, determined by the initiation event and the availability of the Rad52 protein. We discuss the role of the Rad1/Rad10 endonuclease on the initial steps of one-ended invasion crossover and SSA as a function of the location of the initiation event relative to the repeats. We also show that the frequency of recombination between repeats is the same independent of their location (whether on circular plasmids, linear minichromosomes, or natural chromosomes) and have similar RAD52 dependence.     

The Prevention of Repeat-Associated Deletions in Saccharomyces Cerevisiae by Mismatch Repair Depends on Size and Origin of Deletions

We have investigated the effects of mismatch repair on 1- to 61-bp deletions in the yeast Saccharomyces cerevisiae. The deletions are likely to involve unpaired loop intermediates resulting from DNA polymerase slippage. The mutator effects of mutations in the DNA polymerase δ (POL3) gene and the recombinational repair RAD52 gene were studied in combination with mismatch repair defects. The pol3-t mutation increased up to 1000-fold the rate of extended (7-61 bp) but not of 1-bp deletions. In a rad52 null mutant only the 1-bp deletions were increased (12-fold). The mismatch repair mutations pms1, msh2 and msh3 did not affect 31- and 61-bp deletions in the pol3-t but increased the rates of 7- and 1-bp deletions. We propose that loops less than or equal to seven bases generated during replication are subject to mismatch repair by the PMS1, MSH2, MSH3 system and that it cannot act on loops >=31 bases. In contrast to the pol3-t, the enhancement of 1-bp deletions in a rad52 mutant is not altered by a pms1 mutation. Thus, mismatch repair appears to be specific to errors of DNA synthesis generated during semiconservative replication.     

RAD1, an excision repair gene of Saccharomyces cerevisiae, is also involved in recombination.

The RAD1 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of damaged DNA. In this paper, we report our observations on the effect of the RAD1 gene on genetic recombination. Mitotic intrachromosomal and interchromosomal recombination in RAD+, rad1, rad52, and other rad mutant strains was examined. The rad1 deletion mutation and some rad1 point mutations reduced the frequency of intrachromosomal recombination of a his3 duplication, in which one his3 allele is deleted at the 3' end while the other his3 allele is deleted at the 5' end. Mutations in the other excision repair genes, RAD2, RAD3, and RAD4, did not lower recombination frequencies in the his3 duplication. As expected, recombination between the his3 deletion alleles in the duplication was reduced in the rad52 mutant. The frequency of HIS3+ recombinants fell synergistically in the rad1 rad52 double mutant, indicating that the RAD1 and RAD52 genes affect this recombination via different pathways. In contrast to the effect of mutations in the RAD52 gene, mutations in the RAD1 gene did not lower intrachromosomal and interchromosomal recombination between heteroalleles that carry point mutations rather than partial deletions; however, the rad1 delta mutation did lower the frequency of integration of linear plasmids and DNA fragments into homologous genomic sequences. We suggest that RAD1 plays a role in recombination after the formation of the recombinogenic substrate.     

RAD10, an excision repair gene of Saccharomyces cerevisiae, is involved in the RAD1 pathway of mitotic recombination.

The RAD10 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of UV-damaged DNA. We show that the RAD10 gene is also required for mitotic recombination. The rad10 delta mutation lowered the rate of intrachromosomal recombination of a his3 duplication in which one his3 allele has a deletion at the 3' end and the other his3 allele has a deletion at the 5' end (his3 delta 3' his3 delta 5'). The rate of formation of HIS3+ recombinants in the rad10 delta mutant was not affected by the rad1 delta mutation but decreased synergistically in the presence of the rad10 delta mutation in combination with the rad52 delta mutation. These observations indicate that the RAD1 and RAD10 genes function together in a mitotic recombination pathway that is distinct from the RAD52 recombination pathway. The rad10 delta mutation also lowered the efficiency of integration of linear DNA molecules and circular plasmids into homologous genomic sequences. We suggest that the RAD1 and RAD10 gene products act in recombination after the formation of the recombinogenic substrate. The rad1 delta and rad10 delta mutations did not affect meiotic intrachromosomal recombination of the his3 delta 3' his3 delta 5' duplication or mitotic and meiotic recombination of ade2 heteroalleles located on homologous chromosomes.     

C1 inhibitor gene sequence facilitates frameshift mutations.

Mutations disrupting the function or production of C1 inhibitor cause the disease hereditary angioneurotic edema. Patient mutations identified an imperfect inverted repeat sequence that was postulated to play a mechanistic role in the mutations. To test this hypothesis, the inverted repeat was cloned into the chloramphenicol acetyltransferase gene in pBR325 and its mutation rate was studied in four bacterial strains. These strains were selected to assay the effects of recombination and superhelical tension on mutation frequency. Mutations that revert bacteria to chloramphenicol resistance (Cmr) were scored. Both pairs of isogenic strains had reversion frequencies of approximately 10(-8). These rare reversion events in bacteria were most often a frameshift that involved the imperfect inverted repeat with a deletion or a tandem duplication, an event very similar to the human mutations. Increased DNA superhelical tension, which would be expected to enhance cruciform extrusion, did not accentuate mutagenesis. This finding suggests that the imperfect inverted repeat may form a stem-loop structure in the single-stranded DNA created by the duplex DNA melting prior to replication. Models explaining the slippage can be drawn using the lagging strand of the replication fork. In this model, the formation of a stem-loop structure is responsible for bringing the end of the deletion or duplication into close proximity.     

Unrepaired Heteroduplex DNA in Saccharomyces Cerevisiae Is Decreased in Rad1 Rad52-Independent Recombination

A direct repeat recombination assay between SUP4 heteroalleles detects unrepaired heteroduplex DNA (hDNA) as sectored colonies. The frequency of unrepaired heteroduplex is dependent on the mismatch and is highest in a construct that generates C:C or G:G mispairs and lowest in one that generates T:G or C:A mispairs. In addition, unrepaired hDNA increases for all mismatches tested in pms1 mismatch repair-deficient strains. These results support the notion that hDNA is formed across the SUP4 repeats during the recombination event and is then subject to mismatch repair. The effects of various repair and recombination defective mutations on this assay were examined. Unrepaired heteroduplex increases significantly only in rad52 mutant strains. In addition, direct repeat recombination is reduced 2-fold in rad52 mutant strains, while in rad51, rad54, rad55 and rad57 mutants direct repeat recombination is increased 3-4-fold. Mutations in the excision repair gene, RAD1, do not affect the frequency of direct repeat recombination. However, the level of unrepaired heteroduplex is slightly decreased in rad1 mutant strains. Similar to previous studies, rad1 rad52 double mutants show a synergistic reduction in direct repeat recombination (35-fold). Interestingly, unrepaired heteroduplex is reduced 4-fold in the double mutants. Experiments with shortened repeats suggest that the reduction in unrepaired heteroduplex is due to decreased hDNA tract length in the double mutant strain.     

Characterization of the hyperrecombination phenotype of the pol3-t mutation of Saccharomyces cerevisiae

The DNA polymerase delta (Pol3p/Cdc2p) allele pol3-t of Saccharomyces cerevisiae has previously been shown to increase the frequency of deletions between short repeats (several base pairs), between homologous DNA sequences separated by long inverted repeats, and between distant short repeats, increasing the frequency of genomic deletions. We found that the pol3-t mutation increased intrachromosomal recombination events between direct DNA repeats up to 36-fold and interchromosomal recombination 14-fold. The hyperrecombination phenotype of pol3-t was partially dependent on the Rad52p function but much more so on Rad1p. However, in the double-mutant rad1 Delta rad52 Delta, the pol3-t mutation still increased spontaneous intrachromosomal recombination frequencies, suggesting that a Rad1p Rad52p-independent single-strand annealing pathway is involved. UV and gamma-rays were less potent inducers of recombination in the pol3-t mutant, indicating that Pol3p is partly involved in DNA-damage-induced recombination. In contrast, while UV- and gamma-ray-induced intrachromosomal recombination was almost completely abolished in the rad52 or the rad1 rad52 mutant, there was still good induction in those mutants in the pol3-t background, indicating channeling of lesions into the above-mentioned Rad1p Rad52p-independent pathway. Finally, a heterozygous pol3-t/POL3 mutant also showed an increased frequency of deletions and MMS sensitivity at the restrictive temperature, indicating that even a heterozygous polymerase delta mutation might increase the frequency of genetic instability.     

Direction of chromosome rearrangements in Saccharomyces cerevisiae by use of his3 recombinational substrates.

We used the his3 recombinational substrates (his3 fragments) to direct large interchromosomal (translocations) and intrachromosomal (deletions and tandem duplications) rearrangements in the yeast Saccharomyces cerevisiae. In strains completely deleted for the wild-type HIS3 gene, his3 fragments, one containing a deletion of 5' amino acid coding sequences and the other containing a deletion of 3' amino acid coding sequences, were first placed at preselected sites by homologous recombination. His+ revertants that arose via spontaneous mitotic recombination between the two his3 fragments were selected. This strategy was used to direct rearrangements in both RAD52+ and rad52 mutant strains. Translocations occurred in the RAD52+ genetic background and were characterized by orthogonal field alternating gel electrophoresis of yeast chromosomal DNA and by standard genetic techniques. An unexpected translocation was also identified in which HIS3 sequences were amplified. Two types of tandem duplications of the GAL(7, 10, 1) locus were also directed, and one type was not observed in rad52 mutants. Recombination mechanisms are discussed to account for these differences.     

Different frameshift mutation spectra in non-repetitive DNA of MutSalpha- and MutLalpha-deficient fission yeast cells

A frameshift reversion assay has been established for Schizosaccharomyces pombe, which allows detection of deletions and insertions of nucleotides in a non-repetitive DNA sequence. Compared to wild type, frameshift mutation rates were increased in the mismatch repair (MMR) mutants msh2, msh6, mlh1, and pms1, but not in a swi4 strain (defective in the Msh3 homologue). Rates were also elevated in the DNA nuclease-deficient strains rad2 (defective in the FEN-1 homologue) and exo1. In MutSalpha-deficient strains, msh2 and msh6, most of the reversions were 1bp deletions. In contrast, mlh1 and pms1 mutants, defective in MutLalpha, accumulated significantly more 2bp insertions, preferentially of the type CG to (CG)(2). Such duplications were less frequent in double mutants additionally defective in msh2, msh6, rad2, or exo1. Thus, accumulation of (CG)(2) in MutLalpha-deficient strains depends on the presence of MutSalpha, Rad2 and Exo1.     

Insertion elements and deletion formation in a halophilic archaebacterium.

Deletion events that occur spontaneously in 36-kilobase-pair (kbp) plasmid pHH4 from the archaebacterium Halobacterium halobium were investigated. Four different deletion derivatives with sizes ranging from 5.7 to 17 kbp were isolated. Three of these deletion variants derived from pHH4 (pHH6 [17 kbp], pHH7 [16 kbp], and pHH8 [6.3 kbp]), whereas the 5.7-kbp plasmid pHH9 derived from pHH6. Strains containing pHH6, pHH7, or pHH9 each lacked the parental plasmid pHH4, while pHH8 occurred at a 1:1 ratio together with pHH4. Common to all of these plasmids was the 5.7-kbp region of pHH9 DNA. The regions containing the fusion site in the deletion derivatives were investigated and compared with the corresponding area of the parental plasmid. Each deletion occurred exactly at the terminus of an insertion element. In pHH6 and pHH7, a halobacterial insertion element (ISH2) was located at the deletion site. The DNA fused to ISH2 displayed a 7-base-pair (bp) (pHH7) or 10-bp (pHH6) sequence homology to the inverted repeat of ISH2. In the two smaller plasmids, pHH8 and pHH9, an ISH27 element was located at the deletion site. Most likely, all of these smaller plasmids resulted from an intramolecular transposition event. The ISH27 insertion sequence contains a 16-bp terminal inverted repeat and duplicates 5 bp of target DNA during the transposition with the specificity 5'ANNNT3'. Four ISH27 copies were analyzed, and two ISH27 element types were identified that have approximately 85% sequence similarity. The ISH27 insertion elements constitute a family which is related to the ISH51 family characterized for H. volcanii, another halophilic archaebacterium.     

Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break can be repaired by at least two pathways of nonhomologous end joining (NHEJ) that closely resemble events in mammalian cells. In one pathway the chromosome ends are degraded to yield deletions with different sizes whose endpoints have 1 to 6 bp of homology. Alternatively, the 4-bp overhanging 3' ends of HO-cut DNA (5'-AACA-3') are not degraded but can be base paired in misalignment to produce +CA and +ACA insertions. When HO was expressed throughout the cell cycle, the efficiency of NHEJ repair was 30 times higher than when HO was expressed only in G1. The types of repair events were also very different when HO was expressed throughout the cell cycle; 78% of survivors had small insertions, while almost none had large deletions. When HO expression was confined to the G1 phase, only 21% were insertions and 38% had large deletions. These results suggest that there are distinct mechanisms of NHEJ repair producing either insertions or deletions and that these two pathways are differently affected by the time in the cell cycle when HO is expressed. The frequency of NHEJ is unaltered in strains from which RAD1, RAD2, RAD51, RAD52, RAD54, or RAD57 is deleted; however, deletions of RAD50, XRS2, or MRE11 reduced NHEJ by more than 70-fold when HO was not cell cycle regulated. Moreover, mutations in these three genes markedly reduced +CA insertions, while significantly increasing the proportion of both small (-ACA) and larger deletion events. In contrast, the rad5O mutation had little effect on the viability of G1-induced cells but significantly reduced the frequency of both +CA insertions and -ACA deletions in favor of larger deletions. Thus, RAD50 (and by extension XRS2 and MRE11) exerts a much more important role in the insertion-producing pathway of NHEJ repair found in S and/or G2 than in the less frequent deletion events that predominate when HO is expressed only in G1.     

Insights into mutagenesis using Escherichia coli chromosomal lacZ strains that enable detection of a wide spectrum of mutational events

Strand misalignments at DNA repeats during replication are implicated in mutational hotspots. To study these events, we have generated strains carrying mutations in the Escherichia coli chromosomal lacZ gene that revert via deletion of a short duplicated sequence or by template switching within imperfect inverted repeat (quasipalindrome, QP) sequences. Using these strains, we demonstrate that mutation of the distal repeat of a quasipalindrome, with respect to replication fork movement, is about 10-fold higher than the proximal repeat, consistent with more common template switching on the leading strand. The leading strand bias was lost in the absence of exonucleases I and VII, suggesting that it results from more efficient suppression of template switching by 3' exonucleases targeted to the lagging strand. The loss of 3' exonucleases has no effect on strand misalignment at direct repeats to produce deletion. To compare these events to other mutations, we have reengineered reporters (designed by Cupples and Miller 1989) that detect specific base substitutions or frameshifts in lacZ with the reverting lacZ locus on the chromosome rather than an F' element. This set allows rapid screening of potential mutagens, environmental conditions, or genetic loci for effects on a broad set of mutational events. We found that hydroxyurea (HU), which depletes dNTP pools, slightly elevated templated mutations at inverted repeats but had no effect on deletions, simple frameshifts, or base substitutions. Mutations in nucleotide diphosphate kinase, ndk, significantly elevated simple mutations but had little effect on the templated class. Zebularine, a cytosine analog, elevated all classes.     

A switch region inversion contributes to the aberrant rearrangement of a mu immunoglobulin heavy chain gene in MPC-11 cells.

We describe the unique features of an aberrantly rearranged mu immunoglobulin heavy chain gene isolated from MPC-11 cells (a gamma 2b producing Balb/c plasmacytoma). A novel rearrangement has occurred 1.5 Kb 5' of the MPC-11 mu gene (denoted 18b mu) resulting in the deletion of the majority of the repetitive switch region (S mu) and 5' flanking DNA including the Joining (JH) sequences. The remainder (275 bp) of the S mu repeat has undergone a complete sequence inversion. DNA sequences 5' of the inverted S mu sequence do not resemble Variable (VH), Diversity (D), JH or their conserved flanking sequences. A DNA sequence localized 5' of the inverted S mu sequence, (p18b mu-1.4) detects a small family of homologous sequences in Balb/c DNA. The 18b mu-1.4 like sequences lack homology to S mu, exhibit flanking sequence polymorphisms in 5 out of 6 inbred mouse strains and undergo partial or complete deletion in 5 out of 10 plasmacytomas tested. Two 18b mu-1.4 homologous sequences display a higher copy number in C57Bl/6, AL/N and CAL9 mouse strains.     

A novel allele of RAD52 that causes severe DNA repair and recombination deficiencies only in the absence of RAD51 or RAD59.

With the use of an intrachromosomal inverted repeat as a recombination reporter, we have shown that mitotic recombination is dependent on the RAD52 gene, but reduced only fivefold by mutation of RAD51. RAD59, a component of the RAD51-independent pathway, was identified previously by screening for mutations that reduced inverted-repeat recombination in a rad51 strain. Here we describe a rad52 mutation, rad52R70K, that also reduced recombination synergistically in a rad51 background. The phenotype of the rad52R70K strain, which includes weak gamma-ray sensitivity, a fourfold reduction in the rate of inverted-repeat recombination, elevated allelic recombination, sporulation proficiency, and a reduction in the efficiency of mating-type switching and single-strand annealing, was similar to that observed for deletion of the RAD59 gene. However, rad52R70K rad59 double mutants showed synergistic defects in ionizing radiation resistance, sporulation, and mating-type switching. These results suggest that Rad52 and Rad59 have partially overlapping functions and that Rad59 can substitute for this function of Rad52 in a RAD51 rad52R70K strain.     

Three Tn10-associated excision events: relationship to transposition and role of direct and inverted repeats

We describe three related DNA alterations associated with transposon Tn10: precise excision of Tn10, nearly precise excision of Tn10 and precise excision of the nearly precise excision remnant. DNA sequence analysis shows that each of these alterations results in excision of all or part of the Tn10 element, and each involves specific repeat sequences at or near the ends of the element. Furthermore, all three events are structurally analogous: in each case, excision occurs between two short direct-repeat sequences, with resulting deletion of all intervening material plus one copy of the direct repeat; and in all three cases, the direct repeats involved occur at either end of an inverted repeat. Analysis of mutant Tn10 elements and characterization of bacterial host mutations suggest that all three types of excision events occur by pathways that are fundamentally distinct from the pathway(s) for Tn10-promoted transposition and other DNA rearrangements (deletions and inversions) actively promoted by the element. In addition, precise excision and nearly precise excision appear to occur by very closely related or identical pathways; and several lines of evidence suggest that the 1400 bp inverted repeats at the ends of Tn10 may play a structural role in both of these events. The third excision event appears to occur by yet another pathway.     

Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IneW plasmid R388 and comparison with the related IncN plasmid R46

Summary We cloned and sequenced a 402 by DNA segment containing the origin of conjugal transfer (oriT) of the IncW plasmid R388. Progressive deletions from each end of the sequence were assayed for oriT activity. Stepwise reductions in mobilization frequencies, representing the loss of functional elements, correlated with deletion of structural motifs in the sequence. A sequence of 330 by of oriT was sufficient for efficient mobilization. The first 86 by of the sequence contains five tandemly repeated DNA sequences of 11 bp, followed by a 10 by perfect inverted repeat. Deletion of the first 95 by reduced the frequency of transfer by a hundred-fold. The sequence between by 183 and 218 was necessary and sufficient for low frequency mobilization and, thus, it was assumed to contain the nick site. This basis core was cloned as a 60 by segment (from by 176–236) that could be mobilized at low frequency. It includes two inverted repeats and a perfect integration host factor (IHF) consensus binding site. A third functionally important segment in oriT was located between by 260 and 330. The DNA sequence of the oriT of R388 could be aligned with that of the broad-host-range IncN plasmid R46. Moreover, the relative positions of the three inverted repeats are also conserved. Overall sequence similarity was 52%, but was significantly higher in particular regions, whch coincided with the functionally important segments mapped by deletion analysis. Conservation of these segments provided independent support for their essential role in oriT function.     

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.     

Homologous and nonhomologous recombination resulting in deletion: effects of p53 status, microhomology, and repetitive DNA length and orientation

Repetitive DNA elements frequently are precursors to chromosomal deletions in prokaryotes and lower eukaryotes. However, little is known about the relationship between repeated sequences and deletion formation in mammalian cells. We have created a novel integrated plasmid-based recombination assay to investigate repeated sequence instability in human cells. In a control cell line, the presence of direct or inverted repeats did not appreciably influence the very low deletion frequencies (2 x 10(-7) to 9 x 10(-7)) in the region containing the repeat. Similar to what has been observed in lower eukaryotes, the majority of deletions resulted from the loss of the largest direct repeat present in the system along with the intervening sequence. Interestingly, in closely related cell lines that possess a mutant p53 gene, deletion frequencies in the control and direct-repeat plasmids were 40 to 300 times higher than in their wild-type counterparts. However, mutant p53 cells did not preferentially utilize the largest available homology in the formation of the deletion. Surprisingly, inverted repeats were approximately 10,000 times more unstable in all mutant p53 cells than in wild-type cells. Finally, several deletion junctions were marked by the addition of novel bases that were homologous to one of the preexisting DNA ends. Contrary to our expectations, only 6% of deletions in all cell lines could be classified as arising from nonhomologous recombination.