Molecular mechanisms of the formation of DNA double-strand breaks and induction of genomic rearrangements.

The probability that damage occurs in closely opposed sites on complementary DNA strands increases when DNA is heavily modified with mutagenic agents. Enzymatic excision of the opposite lesions produces DNA double-strand breaks which give rise to genomic rearrangements (deletions, insertions, etc.). Plasmid systems were developed for studying chemical lesions leading to double-strand breaks and the fate of broken plasmid molecules within bacterial cells. Deletions result from the base-pairing of fortuitously located direct repeats flanking the DNA broken ends; as a consequence, the latter are joined, while the DNA fragment between the direct repeats is deleted. Genomic rearrangements arise during the repair of the DNA double-strand breaks, and both events are due to similar repair enzymes which maintain the integrity of the DNA primary structure when conditions are not stressful. A number of genomic rearrangements and point mutations seem to be predetermined by the DNA primary structure.     

Stability of an inverted repeat in a human fibrosarcoma cell.

Deletions and rearrangements of DNA sequences within the genome of human cells result in mutations associated with human disease. We have developed a selection system involving a neo gene containing a DNA sequence inserted into the NcoI site that can be used to quantitatively assay deletion of this sequence from the chromosome. The spontaneous deletion from the neo gene of a 122 bp inverted repeat occurred at a rate of 2.1 x 10(-8) to <3.1 x 10(-9) revertants/cell/generation in three different cell lines. Deletion of the 122 bp inverted repeat occurred between 6 bp flanking direct repeats. Spontaneous deletion of a 122 bp non-palindromic DNA sequence flanked by direct repeats was not observed, indicating a rate of deletion of <3.1 x 10(-9) revertants/cell/generation. This result demonstrates that a 122 bp inverted repeat can exhibit a low level of instability in some locations in the chromosome of a human cell line.     

Plasmid R6K DNA replication. II. Direct nucleotide sequence repeats are required for an active gamma-origin

Insertions of the transposable element Tn5 were obtained at a number of sites within the γ origin region of plasmid R6K. A contiguous region of DNA, 240 base-pairs in length, was identified as the functional γ origin of replication on the basis that Tn5 insertions within this region inactivated the origin. This region contains seven, tandemly arranged, 22 base-pair direct repeats. Spontaneous deletion of a Tn5 sequence that is inserted in one of the repeats was accompanied generally by the loss of nucleotide sequences equivalent to one or more of the repeat sequences. Deletion mutants of the γ origin that lack one or two repeats are still functional but a plasmid containing such a mutant origin is not maintained stably. Deletions of nucleotide sequences equivalent to four or more of the direct repeats result in the inactivation of the R6K γ origin.     

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.     

An in Vitro Assay for Frameshift Mutations: Hotspots for Deletions of 1 Bp by Klenow-Fragment Polymerase Share a Consensus DNA Sequence

The fidelity of in vitro DNA synthesis catalyzed by the large fragment of DNA polymerase I was examined. The templates, specifically designed to detect shifts to the +1 or to the -1 reading frame, are composites of M13mp8 and bacteriophage T4 rIIB DNA and were designed to assist in the identification of the types of frameshifts that are the specific consequence of DNA polymerization errors. In vitro polymerization by the Klenow fragment produced only deletions, rather than the mixture of duplications and deletions characteristic of in vivo frameshifts. The most frequent frameshifts were deletions of 1 bp opposite a template purine base. Hotspots for these deletions occurred when the template purine immediately preceded the template sequence TT. The highest mutation frequencies were seen when the TTPu consensus sequence was adjacent to G:C rich sequences in the 3' direction. The nature of the consensus sequence itself distinguishes this 1-bp deletion mechanism from those operating in DNA repeats and attributed to the misalignment of DNA primers during synthesis. Deletions that were larger than 1 or 2 bp isolated after in vitro replication were consistent with the misalignment of the primer. Deletions of 2 bp and complex frameshifts (the replacement of AA by C) were also found. Mechanisms that may account for these mutations are discussed.     

Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated.

HO endonuclease-induced double-strand breaks in Saccharomyces cerevisiae can undergo recombination by two distinct and competing pathways. In a plasmid containing a direct repeat, in which one repeat is interrupted by an HO endonuclease cut site, gap repair yields gene conversions while single-strand annealing produces deletions. Consistent with predictions of the single-strand annealing mechanism, deletion formation is not accompanied by the formation of a reciprocal recombination product. Deletions are delayed 60 min when the distance separating the repeats is increased by 4.4 kb. Moreover, the rate of deletion formation corresponds to the time at which complementary regions become single stranded. Gap repair processes are independent of distance but are reduced in rad52 mutants and in G1-arrested cells.     

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.     

Deletions in Plasmid Pbr322: Replication Slippage Involving Leading and Lagging Strands

We test here whether a class of deletions likely to result from errors during DNA replication arise preferentially during synthesis of either the leading or the lagging DNA strand. Deletions were obtained by reversion of particular insertion mutant alleles of the pBR322 amp gene. The alleles contain insertions of palindromic DNAs bracketed by 9-bp direct repeats of amp sequence; in addition, bp 2 to 5 in one arm of the palindrome form a direct repeat with 4 bp of adjoining amp sequence. Prior work had shown that reversion to Ampr results from deletions with endpoints in the 8- or 4-bp repeat, and that the 4-bp repeats are used preferentially because one of them is in the palindrome. To test the role of leading and lagging strand synthesis in deletion formation, we reversed the direction of replication of the amp gene by inverting the pBR322 replication origin, and also constructed new mutant alleles with a 4-bp repeat starting counterclockwise rather than clockwise of the insertion. In both cases the 4-bp repeats were used preferentially as deletion endpoints. A model is presented in which deletions arise during elongation of the strand that copies the palindrome before the adjoining 4-bp repeat, and in which preferential use of the 4-bp repeats independent of the overall direction of replication implies that deletions arise during syntheses of both leading and lagging strands.     

Deletions within E. coli plasmids carrying yeast rDNA.

Deletions occur in recombinant DNA plasmids that contain yeast ribosomal DNA (rDNA) inserted into the E. coli plasmids pSC101 and pMB9. Deletions within a pMB9 plasmid containing an insert longer than one tandem rDNA repeat apparently are due to homologous recombination because (1) all of the independently derived deletion products of this plasmid lost one complete rDNA repeat (8.6 kb) and retained only a single copy of the segment repeated at the ends of the original insert and (2) deletions were detected only when the insert had terminal redundancy. Deletions also occur within a pSC101 plasmid containing a tandem duplication of a segment (4.7 kb) including both pSC101 DNA and rDNA. Once again these deletions appear to be due to the presence of a duplicated region because all deletion products have lost one complete repeat. Deletions within both of these plasmids took place in both rec+ and recA- host cells, but occurred more frequently in rec+ cells. Oligomerization of the deletion products also occurred in both hosts and was more frequent in rec+ cells.     

Deletions and DNA rearrangements within the transposable DNA element IS2. A model for the creation of palindromic DNA by DNA repair synthesis.

Three derivatives of mutant ga10P-308::IS2-I of Escherichia coli were characterized by DNA sequence analysis. Deletions and DNA sequence rearrangements were observed which apparently were initiated at short A-T rich inverted repeats within IS2. Two of the mutants carried newly synthesized DNA sequences which were inverted copies of already existing IS2 sequences. Thus long stretches with twofold symmetry were formed. It is discussed whether these inverted repeats were formed by DNA repair synthesis which was initiated at the A-T rich palindromes of IS2.     

Ac Induces Homologous Recombination at the Maize P Locus

The maize P gene conditions red phlobaphene pigmentation to the pericarp and cob. Starting from two unstable P alleles which carry insertions of the transposable element Ac, we have derived 51 P null alleles; 47 of the 51 null alleles have a 17-kb deletion which removes the 4.5-kb Ac element and 12.5 kb of P sequences flanking both sides of Ac. The deletion endpoints lie within two 5.2-kb homologous direct repeats which flank the P gene. A P allele which contains the direct repeats, but does not have an Ac insertion between the direct repeats, shows very little sporophytic or gametophytic instability. The apparent frequency of sporophytic mutations was not increased when Ac was introduced in trans. Southern analysis of DNA prepared from the pericarp tissue demonstrates that the deletions can occur premeiotically, in the somatic cells during development of the pericarp. Evidence is presented that the deletions occurred by homologous recombination between the two direct repeats, and that the presence of an Ac element at the P locus is associated with the recombination/deletion. These results add another aspect to the spectrum of activities of Ac: the destabilization of flanking direct repeat sequences.     

Intramolecular transposition by a synthetic IS50 (Tn5) derivative.

We report the formation of deletions and inversions by intramolecular transposition of Tn5-derived mobile elements. The synthetic transposons used contained the IS50 O and I end segments and the transposase gene, a contraselectable gene encoding sucrose sensitivity (sacB), antibiotic resistance genes, and a plasmid replication origin. Both deletions and inversions were associated with loss of a 300-bp segment that is designated the vector because it is outside of the transposon. Deletions were severalfold more frequent than inversions, perhaps reflecting constraints on DNA twisting or abortive transposition. Restriction and DNA sequence analyses showed that both types of rearrangements extended from one transposon end to many different sites in target DNA. In the case of inversions, transposition generated 9-bp direct repeats of target sequences.     

Homologous recombination in bovine papillomavirus shuttlevecter; effect of relative orientation of substrate sequences.

Relative orientation of recombination substrates, neo gene, strongly influenced homologous recombination events in a bovine papillomavirus shuttle vector. Between direct repeats, recombination occurred at a high frequency while between inverted repeats, it was rare. Double strand break near the mutation site increased the recombination frequency between inverted repeats but not between direct repeats. Formation of long heteroduplex as a recombination intermediate may explain this apparently paradoxical phenomenon.     

Electron microscopic mapping of deletions on a streptococcal plasmid carrying extraordinarily long inverted repeats

Deletions Δ101, Δ102, and Δ103 which occurred within the extraordinarily long inverted repeats of the self-ligated large EcoRI fragment of the Streptococcal MLS (macrolides, lincosamides, streptogramin B)-resistance plasmid pSM19035 led to the formation of plasmids pDB101, pDB102, and pDB103. Their molecular lengths were determined by contour length measurements to be 17.8, 17.4, and 13.9 kb, respectively. Electron microscopic examination of self-annealed molecules revealed stem-loop structures with inverted repeats comprising 41 to 91% of the mass of plasmids. Two unique sequences (US₁ and US₂) separated the inverted repeats in the case of pDB101 and pDB103, while in pDB102 the repeats were joined at one end and separated at the other by a unique sequence (US₂). The size of the unique sequence US₂ was identical for all three plasmids, and the location of the resistance determinant was determined by electron microscopic examination of self-annealed molecules of the recombinant plasmid pDB201. Mapping of the deletion termini, accomplished by combining electron microscopic and HindIII restriction data, suggested that deletions may occur at preferential sites.     

Genetic control of intrachromosomal recombination

Intrachromosomal recombination between direct repeats can occur either as gene conversion events, which maintain exactly the number of repeat units, or as deletions, which reduce the number of repeat units. Gene conversions are classical recombination events that utilize the standard chromosome recombination machinery. Spontaneous deletions between direct repeats are generally recA-independent in E. coli and RAD52-independent in S. cerevisiae. This independence from the major recombination genes does not mean that deletions form through a nonrecombinational process. Deletions have been suggested to result from sister chromatid exchange at the replication fork in a recA-independent process. The same type of exchange is proposed to be RAD52-independent in Saccharomyces cerevisiae. RAD52-dependent events encompass all events that involve the initial steps of a recombination reaction, which include strand invasion to form a heteroduplex intermediate.     

The effect of the length of direct repeats and the presence of palindromes on deletion between directly repeated DNA sequences in bacteriophage T7.

The frequency of genetic deletion between directly repeated DNA sequences in bacteriophage T7 was measured as a function of the length of the direct repeat. The non-essential ligase gene (gene 1.3) of bacteriophage T7 was interrupted with pieces of synthetic DNA bracketed by direct repeats of various lengths. Deletion of these 76 bp long inserts was too low to be measured when the direct repeats were less than 6 bp long. However, the frequency of deletion of inserts with longer direct repeats increased exponentially as the length of the repeats increased from 8 to 20 bp. When inverted repeats (palindromes) were designed in the midst of the insert there was essentially no increase in deletion frequency between 10 bp direct repeats. But, the same palindromic sequences increased the deletion frequency between 5 bp direct repeats by at least two orders of magnitude. Thus, in this system homology at the endpoints is a more important determinant of deletion frequency than is the presence of palindromes between the direct repeats.     

Plastome mutator-induced alterations arise in Oenothera chloroplast DNA through template slippage.

The plastome mutator of Oenothera hookeri strain Johansen causes deletions and duplications at target sites defined by direct repeats in the plastid genome. Previous studies characterized the mutations long after they had occurred and could not discriminate between the possibilities that the plastome mutator acted through unequal homologous recombination or template slippage. From the known hotspots, the rRNA spacer in the large inverted repeat was chosen for this study because it contains both direct and indirect repeats. Identical deletions were recovered from independently derived plants; the altered regions were always flanked by direct repeats. The regions in which the deletions occurred have the potential to form secondary structures that would stabilize the intervening sequence. Of the two affected regions, the one with the stronger potential secondary structure was altered more frequently. Because no duplication products or inversions were recovered, it is proposed that the plastome mutator acts through template slippage rather than through a recombination mechanism.     

Illegitimate recombination in a bovine papillomavirus shuttle vector: a high level of site specificity.

Recombination in a bovine papillomavirus shuttle vector carrying direct repeats of Moloney murine leukemia virus LTR sequence was examined. Differently from similar vectors carrying direct repeats of SV40 polyA addition signal or neomycin resistance gene, the vector exhibited no homologous recombination between the repeats. Instead, illegitimate recombination took place. There were two major types of recombination products from the restriction cleavage pattern. The plasmids in independent cellular clones belonging to the same recombination type shared the identical crossover point. Thus, in this plasmid, illegitimate recombination occurred at preferential sites involving exactly the same sequences.     

Creation of a chloroplast microsatellite reporter for detection of replication slippage in Chlamydomonas reinhardtii

Microsatellites are composed of short tandem direct repeats; deletions or duplications of those repeats through the process of replication slippage result in microsatellite instability relative to other genomic loci. Variation in repeat number occurs so frequently that microsatellites can be used for genotyping and forensic analysis. However, an accurate assessment of the rates of change can be difficult because the presence of many repeats makes it difficult to determine whether changes have occurred through single or multiple events. The current study was undertaken to experimentally assess the rates of replication slippage that occur in vivo in the chloroplast DNA of Chlamydomonas reinhardtii. A reporter construct was created in which a stretch of AAAG repeats was inserted into a functional gene to allow changes to be observed when they occurred at the synthetic microsatellite. Restoration of the reading frame occurred through replication slippage in 15 of every million viable cells. Since only one-third of the potential insertion/deletion events would restore the reading frame, the frequency of change could be deduced to be 4.5 x 10(-5). Analysis of the slippage events showed that template slippage was the primary event, resulting in deletions rather than duplications. These findings contrasted with events observed in Escherichia coli during maintenance of the plasmid, where duplications were the rule.