Conservative Intrachromosomal Recombination between Inverted Repeats in Mouse Cells: Association between Reciprocal Exchange and Gene Conversion

Recombination in mammalian cells is thought to involve both reciprocal and nonreciprocal modes of exchange, although rigorous proof is lacking due to the inability to recover all products of an exchange. To investigate further the relationship between these modes of exchange, we have analyzed intrachromosomal recombination between duplicated herpes simplex virus thymidine kinase (HSV tk) mutant alleles arranged as inverted repeats in cultured mouse L cells. In crosses between inverted repeats, a single intrachromatid reciprocal exchange leads to inversion of the sequence between the crossover sites and recovery of both genes involved in the event. The majority of recombinant products do not display such inversion and are thus consistent with a nonreciprocal mode of recombination (gene conversion). The remaining products display the sequence inversion predicted for intrachromatid reciprocal exchange. In light of the fact that intrachromatid exchanges occur, the rarity of intrachromatid double reciprocal exchanges strengthens the interpretation that the majority of events in this and previous investigations involve gene conversion. Furthermore, in accord with prediction, one-third of the reciprocal recombinants (inversions) display associated gene conversion. This association suggests that reciprocal and nonreciprocal modes of exchange are mechanistically related in mammalian cells. Finally, the occurrence of inversion recombinants suggests that intrachromosomal recombination can be a conservative (nondestructive) process.     

Yeast Intrachromosomal Recombination: Long Gene Conversion Tracts Are Preferentially Associated with Reciprocal Exchange and Require the Rad1 and Rad3 Gene Products

A yeast intrachromosomal recombination system based on an inverted repeat has been designed to examine mitotic gene conversion tract length and the association of crossing over with gene conversion as a function of the conversion tract length. Short conversion tracts are found to be preferentially noncrossover while conversion tracts longer than 1.16 kb show a 50% association with crossover. Mutation in the excision repair gene RAD1 leads to a reduction in conversion tracts of at least 1.16 kb and a reduction in crossovers associated with conversion, regardless of the length of the conversion tract. Mutation in the excision repair gene RAD3, which encodes a DNA helicase, also leads to a reduction in conversion tracts of at least 1.16 kb, but has no effect on the frequency of associated crossovers. The roles of RAD1 and RAD3 in recombination are discussed.     

Distance-Independence of Mitotic Intrachromosomal Recombination in Saccharomyces Cerevisiae

Many genetic studies have shown that the frequency of homologous recombination depends largely on the distance in which recombination can occur. We have studied the effect of varying the length of duplicated sequences on the frequency of mitotic intrachromosomal recombination in Saccharomyces cerevisiae. We find that the frequency of recombination resulting in the loss of one of the repeats and the intervening sequences reaches a plateau when the repeats are short. In addition, the frequency of recombination to correct a point mutation contained in one of these repeats is not proportional to the size of the duplication but rather depends dramatically on the location of the mutation within the repeated sequences. However, the frequency of mitotic interchromosomal reciprocal recombination is dependent on the distance separating the markers. The difference in the response of intrachromosomal and interchromosomal mitotic recombination to increasing lengths of homology may indicate there are different rate-limiting steps for recombination in these two cases. These findings have important implications for the maintenance and evolution of duplicated sequences.     

Size Of Gene Specific Inverted Repeat - Dependent Gene Deletion In Saccharomyces cerevisiae

We describe here an approach for rapidly producing scar-free and precise gene deletions in S. cerevisiae with high efficiency. Preparation of the disruption gene cassette in this approach was simply performed by overlap extension-PCR of an invert repeat of a partial or complete sequence of the targeted gene with URA3. Integration of the prepared disruption gene cassette to the designated position of a target gene leads to the formation of a mutagenesis cassette within the yeast genome, which consists of a URA3 gene flanked by the targeted gene and its inverted repeat between two short identical direct repeats. The inherent instability of the inverted sequences in close proximity facilitates the self-excision of the entire mutagenesis cassette deposited in the genome and promotes homologous recombination resulting in a seamless deletion via a single transformation. This rapid assembly circumvents the difficulty during preparation of disruption gene cassettes composed of two inverted repeats of the URA3, which requires the engineering of unique restriction sites for subsequent digestion and T4 DNA ligation in vitro. We further identified that the excision of the entire mutagenesis cassette flanked by two DRs in the transformed S. cerevisiae is dependent on the length of the inverted repeat of which a minimum of 800 bp is required for effective gene deletion. The deletion efficiency improves with the increase of the inverted repeat till 1.2 kb. Finally, the use of gene-specific inverted repeats of target genes enables simultaneous gene deletions. The procedure has the potential for application on other yeast strains to achieve precise and efficient removal of gene sequences.     

Role of Double-Strand Break End-Tethering during Gene Conversion in Saccharomyces cerevisiae

Correct repair of DNA double-strand breaks (DSBs) is critical for maintaining genome stability. Whereas gene conversion (GC)-mediated repair is mostly error-free, repair by break-induced replication (BIR) is associated with non-reciprocal translocations and loss of heterozygosity. We have previously shown that a Recombination Execution Checkpoint (REC) mediates this competition by preventing the BIR pathway from acting on DSBs that can be repaired by GC. Here, we asked if the REC can also determine whether the ends that are engaged in a GC-compatible configuration belong to the same break, since repair involving ends from different breaks will produce potentially deleterious translocations. We report that the kinetics of repair are markedly delayed when the two DSB ends that participate in GC belong to different DSBs (termed Trans) compared to the case when both DSB ends come from the same break (Cis). However, repair in Trans still occurs by GC rather than BIR, and the overall efficiency of repair is comparable. Hence, the REC is not sensitive to the “origin” of the DSB ends. When the homologous ends for GC are in Trans, the delay in repair appears to reflect their tethering to sequences on the other side of the DSB that themselves recombine with other genomic locations with which they share sequence homology. These data support previous observations that the two ends of a DSB are usually tethered to each other and that this tethering facilitates both ends encountering the same donor sequence. We also found that the presence of homeologous/repetitive sequences in the vicinity of a DSB can distract the DSB end from finding its bona fide homologous donor, and that inhibition of GC by such homeologous sequences is markedly increased upon deleting Sgs1 but not Msh6.     

Instability of a Plasmid-Borne Inverted Repeat in Saccharomyces Cerevisiae

Inverted repeated DNA sequences are common in both prokaryotes and eukaryotes. We found that a plasmid-borne 94 base-pair inverted repeat (a perfect palindrome of 47 bp) containing a poly GT sequence is unstable in S. cerevisiae, with a minimal deletion frequency of about 10(-4)/mitotic division. Ten independent deletions had identical end points. Sequence analysis indicated that all deletions were the result of a DNA polymerase slippage event (or a recombination event) involving a 5-bp repeat (5' CGACG 3') that flanked the inverted repeat. The deletion rate and the types of deletions were unaffected by the rad52 mutation. Strains with the pms1 mutation had a 10-fold elevated frequency of instability of the inverted repeat. The types of sequence alterations observed in the pms1 background, however, were different than those seen in either the wild-type or rad52 genetic backgrounds.     

A Two-Pathway Analysis of Meiotic Crossing Over and Gene Conversion in Saccharomyces cerevisiae

Several apparently paradoxical observations regarding meiotic crossing over and gene conversion are readily resolved in a framework that recognizes the existence of two recombination pathways that differ in mismatch repair, structures of intermediates, crossover interference, and the generation of noncrossovers. One manifestation of these differences is that simultaneous gene conversion on both sides of a recombination-initiating DNA double-strand break (“two-sidedness”) characterizes only one of the two pathways and is promoted by mismatch repair. Data from previous work are analyzed quantitatively within this framework, and a molecular model for meiotic double-strand break repair based on the concept of sliding D-loops is offered as an efficient scheme for visualizing the salient results from studies of crossing over and gene conversion, the molecular structures of recombination intermediates, and the biochemical competencies of the proteins involved.EUKARYOTES transit from the diplophase to the haplophase via meiosis, which is associated with a number of interrelated processes, including crossing over and gene conversion. These processes involve meiosis-specific, programmed DNA double-strand breaks (DSBs) and their repair (DSBr). DSBr, in turn, is associated with mismatched base pairs and their rectification, referred to as “mismatch repair” or MMR (Bishop et al. 1987). Current efforts to accommodate both the genetic and molecular phenomena associated with meiotic DSBr in yeast (Saccharomyces cerevisiae) have been thoroughly reviewed (e.g., Hollingsworth and Brill 2004; Hoffmann and Borts 2004; Surtees et al. 2004; Hunter 2007; Berchowitz and Copenhaver 2010), but none of the reviews commits to an overall picture with quantitative predictions. This work aims to remedy that lack. Specifically, we have made use of salient published studies to develop, step-by-step, a comprehensive model of meiotic DSBr and MMR. The main features of this model are summarized in

Telomere Recombination Accelerates Cellular Aging in Saccharomyces cerevisiae

Telomeres are nucleoprotein structures located at the linear ends of eukaryotic chromosomes. Telomere integrity is required for cell proliferation and survival. Although the vast majority of eukaryotic species use telomerase as a primary means for telomere maintenance, a few species can use recombination or retrotransposon-mediated maintenance pathways. Since Saccharomyces cerevisiae can use both telomerase and recombination to replicate telomeres, budding yeast provides a useful system with which to examine the evolutionary advantages of telomerase and recombination in preserving an organism or cell under natural selection. In this study, we examined the life span in telomerase-null, post-senescent type II survivors that have employed homologous recombination to replicate their telomeres. Type II recombination survivors stably maintained chromosomal integrity but exhibited a significantly reduced replicative life span. Normal patterns of cell morphology at the end of a replicative life span and aging-dependent sterility were observed in telomerase-null type II survivors, suggesting the type II survivors aged prematurely in a manner that is phenotypically consistent with that of wild-type senescent cells. The shortened life span of type II survivors was extended by calorie restriction or TOR1 deletion, but not by Fob1p inactivation or Sir2p over-expression. Intriguingly, rDNA recombination was decreased in type II survivors, indicating that the premature aging of type II survivors was not caused by an increase in extra-chromosomal rDNA circle accumulation. Reintroduction of telomerase activity immediately restored the replicative life span of type II survivors despite their heterogeneous telomeres. These results suggest that telomere recombination accelerates cellular aging in telomerase-null type II survivors and that telomerase is likely a superior telomere maintenance pathway in sustaining yeast replicative life span.     

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.     

P-Element-Induced Male Recombination and Gene Conversion in Drosophila

A P-element insertion flanked by 13 restriction fragment length polymorphism (RFLP) marker sites was used to examine male recombination and gene conversion at an autosomal site. The great majority of crossovers on chromosome arm 2R occurred within the 4-kb region containing the P element and RFLP sites. Of the 128 recombinants analyzed, approximately two-thirds carried duplications or deletions flanking the P element. These rearrangements are described in more detail in the accompanying report. In a parallel experiment, we examined 91 gene conversion tracts resulting from excision of the same autosomal P element. We found the average tract length was 1463 bp, which is essentially the same as found previously at the white locus. The distribution of conversion tract endpoints was indistinguishable from the distribution of crossover points among the nonrearranged male recombinants. Most recombination events can be explained by the ``hybrid element insertion' model, but, for those lacking a duplication or deletion, a second step involving double-strand gap repair must be postulated to explain the distribution of crossover points.     

Analysis of a Recombination Hotspot for Gene Conversion Occurring at the His2 Gene of Saccharomyces Cerevisiae

The properties of gene conversion as measured in fungi that generate asci containing all the products of meiosis imply that meiotic recombination initiates at specific sites. The HIS2 gene of Saccharomyces cerevisiae displays a high frequency of gene conversion, indicating that it is a recombination hotspot. The HIS2 gene was cloned and sequenced, and the cloned DNA was used to make several different types of alterations in the yeast chromosome by transformation; these alterations were used to determine the location of the sequences necessary for the high levels of meiotic conversion observed at HIS2. Previous work indicated that the gene conversion polarity gradient is high at the 3' end of the gene, and that the promoter of the gene is not necessary for the high frequency of conversion observed. Data presented here suggest that at least some of the sequences necessary for high levels of conversion at HIS2 are located over 700 bp downstream of the end of the coding region, extend over (at least) several hundred base pairs, and may be quite complex, perhaps involving chromatin structure. Additional data indicate that multiple single base heterologies within a 1-kb interval contribute little to the frequency of gene conversion. This contrasts with other reports about the role of heterologies at the MAT locus.     

Intrachromosomal Gene Conversion and the Maintenance of Sequence Homogeneity among Repeated Genes

Intrachromosomal gene conversion is the non-reciprocal transfer of information between a pair of repeated genes on a single chromosome. This process produces eventual sequence homogeneity within a family of repeated genes. An evolutionary model for a single chromosome lineage was formulated and analyzed. Expressions were derived for the fixation probability, mean time to fixation or loss, and mean conditional fixation time for a variant repeat with an arbitrary initial frequency. It was shown that a small conversional advantage or disadvantage for the variant repeat (higher or lower probability of producing two variant genes by conversion than two wild-type genes) can have a dramatic effect on the probability of fixation. The results imply that intrachromosomal gene conversion can act sufficiently rapidly to be an important mechanism for maintaining sequence homogeneity among repeated genes.     

Meiotic Gene Conversion and Crossing over between Dispersed Homologous Sequences Occurs Frequently in Saccharomyces cerevisiae

We have examined meiotic recombination between two defined leu2 heteroalleles present at the normal LEU2 locus and in leu2-containing plasmids inserted at four other genomic locations. In diploids where the two leu2 markers were present at allelic locations on parental homologs, the frequency of Leu2+ spores varied 38-fold, in a location-dependent manner. These results indicate that recombination in a genetic interval can be modulated by sequences at least 2.7 kb outside that interval. Leu2+ meiotic segregants were also recovered from diploids where LEU2 was marked with one heteroallele, and the other leu2 heteroallele was inserted at another genomic location. These products of ectopic interactions, between dispersed copies of leu2 sharing only 2.2 kb of homology, were recovered at a frequency comparable to that observed in corresponding allelic crosses. This high frequency of ectopic meiotic recombination was observed in crosses where both recombining partners could potentially pair with sequences at an allelic position. In addition, a significant fraction (22-50%) of these ectopic recombinants were associated with crossing over of flanking sequences.     

High-Resolution Mapping of Two Types of Spontaneous Mitotic Gene Conversion Events in Saccharomyces cerevisiae

Gene conversions and crossovers are related products of the repair of double-stranded DNA breaks by homologous recombination. Most previous studies of mitotic gene conversion events have been restricted to measuring conversion tracts that are <5 kb. Using a genetic assay in which the lengths of very long gene conversion tracts can be measured, we detected two types of conversions: those with a median size of ∼6 kb and those with a median size of >50 kb. The unusually long tracts are initiated at a naturally occurring recombination hotspot formed by two inverted Ty elements. We suggest that these long gene conversion events may be generated by a mechanism (break-induced replication or repair of a double-stranded DNA gap) different from the short conversion tracts that likely reflect heteroduplex formation followed by DNA mismatch repair. Both the short and long mitotic conversion tracts are considerably longer than those observed in meiosis. Since mitotic crossovers in a diploid can result in a heterozygous recessive deleterious mutation becoming homozygous, it has been suggested that the repair of DNA breaks by mitotic recombination involves gene conversion events that are unassociated with crossing over. In contrast to this prediction, we found that ∼40% of the conversion tracts are associated with crossovers. Spontaneous mitotic crossover events in yeast are frequent enough to be an important factor in genome evolution.     

Reciprocal translocations in Saccharomyces cerevisiae formed by nonhomologous end joining

Reciprocal translocations are common in cancer cells, but their creation is poorly understood. We have developed an assay system in Saccharomyces cerevisiae to study reciprocal translocation formation in the absence of homology. We induce two specific double-strand breaks (DSBs) simultaneously on separate chromosomes with HO endonuclease and analyze the subsequent chromosomal rearrangements among surviving cells. Under these conditions, reciprocal translocations via nonhomologous end joining (NHEJ) occur at frequencies of approximately 2-7 x 10(-5)/cell exposed to the DSBs. Yku80p is a component of the cell's NHEJ machinery. In its absence, reciprocal translocations still occur, but the junctions are associated with deletions and extended overlapping sequences. After induction of a single DSB, translocations and inversions are recovered in wild-type and rad52 strains. In these rearrangements, a nonrandom assortment of sites have fused to the DSB, and their junctions show typical signs of NHEJ. The sites tend to be between open reading frames or within Ty1 LTRs. In some cases the translocation partner is formed by a break at a cryptic HO recognition site. Our results demonstrate that NHEJ-mediated reciprocal translocations can form in S. cerevisiae as a consequence of DSB repair.