L29M] substitution in the interface of subunit-subunit interactions enhances Escherichia coli RecA protein properties important for its recombinogenic activity.

Genetic analysis of RecA protein chimeras and their ancestors, RecAEc (from Escherichia coli) and RecAPa (Pseudomonas aeruginosa) had allowed us to place these proteins with respect to their recombinogenic activities in the following order: RecAPa>RecAX21>RecAX20=RecAEc. While RecAX20 differs from RecAEc in five amino acid residues with two substitutions ([S25A] and [I26V]) at the interface of subunit interactions in the RecA polymer, RecAX20 and RecAX21 differ only by a single substitution [L29M] present at the interface. Here, we present an analysis of the biochemical properties considered important for the recombinogenic activity of all four RecA proteins. While RecAX20 was very similar to RecAEc by all activities analysed, RecAX21 differed from RecAEc in several respects. These differences included an increased affinity for double-stranded DNA, a more active displacement of SSB protein from single-stranded DNA (ssDNA), a decreased end-dependent RecAX21 protein dissociation from a presynaptic complex, and a greater accumulation of intermediate products relative to the final product in the strand-exchange reaction. RecAPa was more tolerant than RecAX21 only to the end-dependent RecA protein dissociation. In addition, RecAPa was more resistant to temperature and salt concentrations in its ability to form a presynaptic RecAPa::ATP::ssDNA filament. Calculations of conformational energy revealed that the [L29M] substitution in RecAX21 polymer caused an increase in its flexibility. This led us to conclude that even a small change in the flexibility of the RecA presynaptic complex could profoundly affect its recombinogenic properties.     

Distinguishing characteristics of hyperrecombinogenic RecA protein from Pseudomonas aeruginosa acting in Escherichia coli

In Escherichia coli, a relatively low frequency of recombination exchanges (FRE) is predetermined by the activity of RecA protein, as modulated by a complex regulatory program involving both autoregulation and other factors. The RecA protein of Pseudomonas aeruginosa (RecA(Pa)) exhibits a more robust recombinase activity than its E. coli counterpart (RecA(Ec)). Low-level expression of RecA(Pa) in E. coli cells results in hyperrecombination (an increase of FRE) even in the presence of RecA(Ec). This genetic effect is supported by the biochemical finding that the RecA(Pa) protein is more efficient in filament formation than RecA K72R, a mutant protein with RecA(Ec)-like DNA-binding ability. Expression of RecA(Pa) also partially suppresses the effects of recF, recO, and recR mutations. In concordance with the latter, RecA(Pa) filaments initiate recombination equally from both the 5' and 3' ends. Besides, these filaments exhibit more resistance to disassembly from the 5' ends that makes the ends potentially appropriate for initiation of strand exchange. These comparative genetic and biochemical characteristics reveal that multiple levels are used by bacteria for a programmed regulation of their recombination activities.     

Conjugational hyperrecombination achieved by derepressing the LexA regulon,altering the properties of RecA protein and inactivating mismatch repair in Escherichia coli K-12

The frequency of recombinational exchanges (FRE) that disrupt co-inheritance of transferred donor markers in Escherichia coli Hfr by F(-) crosses differs by up to a factor of two depending on physiological factors and culture conditions. Under standard conditions we found FRE to be 5.01 +/- 0.43 exchanges per 100-min units of DNA length for wild-type strains of the AB1157 line. Using these conditions we showed a cumulative effect of various mutations on FRE. Constitutive SOS expression by lexA gene inactivation (lexA71::Tn5) and recA gene mutation (recA730) showed, respectively, approximately 4- and 7-fold increases of FRE. The double lexA71 recA730 combination gave an approximately 17-fold increase in FRE. Addition of mutS215::Tn10, inactivating the mismatch repair system, to the double lexA recA mutant increased FRE to approximately 26-fold above wild-type FRE. Finally, we showed that another recA mutation produced as much SOS expression as recA730 but increased FRE only 3-fold. We conclude that three factors contribute to normally low FRE under standard conditions: repression of the LexA regulon, the properties of wild-type RecA protein, and a functioning MutSHL mismatch repair system. We discuss mechanisms by which the lexA, recA, and mutS mutations may elevate FRE cumulatively to obtain hyperrecombination.     

Hyper-recombinogenic RecA protein from Pseudomonas aeruginosa with enhanced activity of its primary DNA binding site

According to one prominent model, each protomer in the activated nucleoprotein filament of homologous recombinase RecA possesses two DNA-binding sites. The primary site binds (1) single-stranded DNA (ssDNA) to form presynaptic complex and (2) the newly formed double-stranded (ds) DNA whereas the secondary site binds (1) dsDNA of a partner to initiate strand exchange and (2) the displaced ssDNA following the strand exchange. RecA protein from Pseudomonas aeruginosa (RecAPa) promotes in Escherichia coli hyper-recombination in an SOS-independent manner. Earlier we revealed that RecAPa rapidly displaces E.coli SSB protein (SSB-Ec) from ssDNA to form presynaptic complex. Here we show that this property (1) is based on increased affinity of ssDNA for the RecAPa primary DNA binding site while the affinity for the secondary site remains similar to that for E.coli RecA, (2) is not specific for SSB-Ec but is also observed for SSB protein from P.aeruginosa that, in turn, predicts a possibility of enhanced recombination repair in this pathogenic bacterium.     

Enzymatic control of homologous recombination and hyperrecombination in Escherichia coli

The RecA protein is a central homologous recombination enzyme in the bacterial cell. Forming a right-handed filament on ssDNA, RecA provides for a homology search between two DNA molecules and homologous strand exchange. RecA protects the cell from ionizing radiation and UV light and is capable of completing recombination during normal cell growth. Several mutant and natural RecA forms have a higher recombination potential in vitro and in vivo as compared with the wild-type Escherichia coli RecA, causing hyperrecombination. Recombinational hyperactivity of RecA depends to a great extent on the filamentation dynamics and DNA transferase properties, which may depend not only on specific amino acid substitutions in RecA, but also by defects in cell enzymatic machinery, including RecO, RecR, RecF, RecX, DinI, SSB, and PsiB. The functions of these proteins are currently known at the molecular level, while their roles in hyperrecombination are still incompletely understood. An increase in recombination in vivo is not always advantageous for the cell and is therefore limited by various mechanisms. In addition to the limitations imposed by cell enzymatic machinery, genomic rearrangements aimed at inhibiting the expression of hyperactive RecA are fixed through cell generations via selection against hyperrecombination. The mechanisms regulating hyperactive RecA forms in several model systems are considered.     

Gene conversion tracts associated with crossovers in Rhizobium etli

Gene conversion has been defined as the nonreciprocal transfer of information between homologous sequences. Despite its broad interest for genome evolution, the occurrence of this mechanism in bacteria has been difficult to ascertain due to the possible occurrence of multiple crossover events that would mimic gene conversion. In this work, we employ a novel system, based on cointegrate formation, to isolate gene conversion events associated with crossovers in the nitrogen-fixing bacterium Rhizobium etli. In this system, selection is applied only for cointegrate formation, with gene conversions being detected as unselected events. This minimizes the likelihood of multiple crossovers. To track the extent and architecture of gene conversions, evenly spaced nucleotide changes were made in one of the nitrogenase structural genes (nifH), introducing unique sites for different restriction endonucleases. Our results show that (i) crossover events were almost invariably accompanied by a gene conversion event occurring nearby; (ii) gene conversion events ranged in size from 150 bp to 800 bp; (iii) gene conversion events displayed a strong bias, favoring the preservation of incoming sequences; (iv) even small amounts of sequence divergence had a strong effect on recombination frequency; and (v) the MutS mismatch repair system plays an important role in determining the length of gene conversion segments. A detailed analysis of the architecture of the conversion events suggests that multiple crossovers are an unlikely alternative for their generation. Our results are better explained as the product of true gene conversions occurring under the double-strand break repair model for recombination.     

Meiotic Crossing over between Nonhomologous Chromosomes Affects Chromosome Segregation in Yeast

Meiotic recombination between artificial repeats positioned on nonhomologous chromosomes occurs efficiently in the yeast Saccharomyces cerevisiae. Both gene conversion and crossover events have been observed, with crossovers yielding reciprocal translocations. In the current study, 5.5-kb ura3 repeats positioned on chromosomes V and XV were used to examine the effect of ectopic recombination on meiotic chromosome segregation. Ura(+) random spores were selected and gene conversion vs. crossover events were distinguished by Southern blot analysis. Approximately 15% of the crossover events between chromosomes V and XV were associated with missegregation of one of these chromosomes. The missegregation was manifest as hyperploid spores containing either both translocations plus a normal chromosome, or both normal chromosomes plus one of the translocations. In those cases where it could be analyzed, missegregation occurred at the first meiotic division. These data are discussed in terms of a model in which ectopic crossovers compete efficiently with normal allelic crossovers in directing meiotic chromosome segregation.     

Five RecA-like proteins of Schizosaccharomyces pombe are involved in meiotic recombination

The genome of Schizosaccharomyces pombe contains five genes that code for proteins with sequence similarity to the Escherichia coli recombination protein RecA: rad51+, rhp55+, rhp57+, rlp1+, and dmc1+. We analyzed the effect of deletion of each of these genes on meiotic recombination and viability of spores. Meiotic recombination levels were different from wild type in all recA-related mutants in several genetic intervals, suggesting that all five RecA homologs of S. pombe are required for normal levels of meiotic recombination. Spore viability was reduced in rad51, rhp55, and rhp57 mutants, but not in rlp1 and dmc1. It is argued that reduction of crossover is not the only cause for the observed reduction of spore viability. Analysis of double and triple mutants revealed that Rad51 and Dmc1 play major and partially overlapping roles in meiotic recombination, while Rhp55, Rhp57, and Rlp1 play accessory roles. Remarkably, deletion of Rlp1 decreases the frequency of intergenic recombination (crossovers), but increases intragenic recombination (gene conversion). On the basis of our results, we present a model for the involvement of five RecA-like proteins of S. pombe in meiotic recombination and discuss their respective roles.     

Use of a Chromosomal Inverted Repeat to Demonstrate That the Rad51 and Rad52 Genes of Saccharomyces Cerevisiae Have Different Roles in Mitotic Recombination

An intrachromosomal recombination assay that monitors events between alleles of the ade2 gene oriented as inverted repeats was developed. Recombination to adenine prototrophy occurred at a rate of 9.3 X 10(-5)/cell/generation. Of the total recombinants, 50% occurred by gene conversion without crossing over, 35% by crossover and 15% by crossover associated with conversion. The rate of recombination was reduced 3,000-fold in a rad52 mutant, but the distribution of residual recombination events remained similar to that seen in the wild type strain. In rad51 mutants the rate of recombination was reduced only 4-fold. In this case, gene conversion events unassociated with a crossover were reduced 18-fold, whereas crossover events were reduced only 2.5-fold. A rad51 rad52 double mutant strain showed the same reduction in the rate of recombination as the rad52 mutant, but the distribution of events resembled that seen in rad51. From these observations it is concluded that (i) RAD52 is required for high levels of both gene conversions and reciprocal crossovers, (ii) that RAD51 is not required for intrachromosomal crossovers, and (iii) that RAD51 and RAD52 have different functions, or that RAD52 had functions in addition to those of the Rad51/Rad52 protein complex.     

Genetic and Molecular Analysis of Recombination Events in Saccharomyces Cerevisiae Occurring in the Presence of the Hyper-Recombination Mutation Hpr1

The hyper-recombination mutation hpr1 specifically increases mitotic intrachromatid crossovers, with no effect on other mitotic recombination events such as unequal sister chromatid exchange and plasmid-chromosome recombination, and no effect on meiotic recombination and a lesser effect on intrachromosomal gene conversion. The excision repair RAD1 gene is partially required for the expression on the hpr1 phenotype. The simplest hypothesis to account for some of the hpr1 stimulated recombination events is that a heteroduplex DNA intermediate and localized gene conversion are involved. hpr1 stimulated crossover events are independent of intrachromosomal gene conversion events stimulated by the hyper-gene conversion mutation hpr5. This result suggests that different intrachromosomal recombination processes are affected in each mutant strain. We propose that HPR1 may function to inhibit intrachromatid crossovers.     

Gene Conversion Alone Accounts for More than 90% of Recombination Events at the Am Locus of Neurospora Crassa

We have used closely flanking molecular markers located ~4 kb distal and 6 kb proximal of the am locus to investigate the incidence of crossover events associated with the generation of prototrophic recombinants in a cross heteroallelic am(1) am(6). Ninety-three percent of prototrophs were generated by events that did not recombine the molecular markers, indicating that simple conversion accounts for the formation of most prototrophs and that associated crossovers are much less frequent (~0.07) than estimated previously using more distant flanking markers. This suggests that conversion and crossing over during meiosis may arise from distinct mechanisms or that if, as is widely supposed, conversion and crossing over result from alternate modes of resolution of Holliday junctions then, at least for the am locus of Neurospora, the mode of resolution is strongly biased in favor of retaining the parental association of flanking sequences. Because estimates of the association of conversion and crossing over based on more distant gene markers are similar for yeast and Neurospora (~0.35), our observation may have general significance.     

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.     

Meiotic crossing-over: obligation and interference

During meiosis, crossing-over--the exchange of genetic material between maternal and paternal chromosomes--is stringently controlled to restrict the number of crossovers per chromosome pair. In this issue of Cell, Martini et al., (2006) report that the reduction of crossover-initiating events does not result in fewer crossovers. These results have important implications for our understanding of crossover control.     

Studies on crossover-specific mutants and the distribution of crossing over in Drosophila females

In Drosophila females, the majority of recombination events do not become crossovers and those that do occur are nonrandomly distributed. Furthermore, a group of Drosophila mutants specifically reduce crossing over, suggesting that crossovers depend on different gene products than noncrossovers. In mei-218 mutants, crossing over is reduced by approximately 90% while noncrossovers and the initiation of recombination remain unchanged. Importantly, the residual crossovers have a more random distribution than wild-type. It has been proposed that mei-218 has a role in establishing the crossover distribution by determining which recombination sites become crossovers. Surprisingly, a diverse group of genes, including those required for double strand break (DSB) formation or repair, have an effect on crossover distribution. Not all of these mutants, however, have a crossover-specific defect like mei-218 and it is not understood why some crossover-defective mutants alter the distribution of crossovers. Intragenic recombination experiments suggest that mei-218 is required for a molecular transition of the recombination intermediate late in the DSB repair pathway. We propose that the changes in crossover distribution in some crossover-defective mutants are a secondary consequence of the crossover reductions. This may be the activation of a regulatory system that ensures at least one crossover per chromosome, and which compensates for an absence of crossovers by attempting to generate them at random locations.     

Distinct functions of MLH3 at recombination hot spots in the mouse

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

Two meiotic crossover classes cohabit in Arabidopsis: one is dependent on MER3,whereas the other one is not

BACKGROUND: Crossovers are essential for the completion of meiosis. Recently, two pathways of crossover formation have been identified on the basis of distinct genetic controls. In one pathway, crossover inhibits the occurrence of another such event in a distance-dependent manner. This phenomenon is known as interference. The second kind of crossover is insensitive to interference. The two pathways function independently in budding yeast. Only interference-insensitive crossovers occur in Schizosaccharomyces pombe. In contrast, only interference-sensitive crossovers occur in Caenorabditis elegans. The situation in mammals and plants remains unclear. Mer3 is one of the genes shown to be required for the formation of interference-sensitive crossovers in Saccharomyces cerevisiae. RESULTS: To unravel the crossover status in the plant Arabidopsis thaliana, we investigated the role of the A. thaliana MER3 gene through the characterization of a series of allelic mutants. All mer3 mutants showed low levels of fertility and a significant decrease (about 75%) but not a total disappearance of meiotic crossovers, with the number of recombination events initiated in the mutants being similar to that in the wild-type. Genetic analyses showed that the residual crossovers in mer3 mutants did not display interference in one set of adjacent intervals. CONCLUSIONS: Mutation in MER3 in Arabidopsis appeared to be specific to recombination events resulting in interference-sensitive crossovers. Thus, MER3 function is conserved from yeast to plants and may exist in other metazoans. Arabidopsis therefore has at least two pathways for crossover formation, one giving rise to interference-sensitive crossover and the other to independently distributed crossovers.     

Meiotic,genomic and evolutionary properties of crossover distribution in Drosophila yakuba

The number and location of crossovers across genomes are highly regulated during meiosis, yet the key components controlling them are fast evolving, hindering our understanding of the mechanistic causes and evolutionary consequences of changes in crossover rates. Drosophila melanogaster has been a model species to study meiosis for more than a century, with an available high-resolution crossover map that is, nonetheless, missing for closely related species, thus preventing evolutionary context. Here, we applied a novel and highly efficient approach to generate whole-genome high-resolution crossover maps in D. yakuba to tackle multiple questions that benefit from being addressed collectively within an appropriate phylogenetic framework, in our case the D. melanogaster species subgroup. The genotyping of more than 1,600 individual meiotic events allowed us to identify several key distinct properties relative to D. melanogaster. We show that D. yakuba, in addition to higher crossover rates than D. melanogaster, has a stronger centromere effect and crossover assurance than any Drosophila species analyzed to date. We also report the presence of an active crossover-associated meiotic drive mechanism for the X chromosome that results in the preferential inclusion in oocytes of chromatids with crossovers. Our evolutionary and genomic analyses suggest that the genome-wide landscape of crossover rates in D. yakuba has been fairly stable and captures a significant signal of the ancestral crossover landscape for the whole D. melanogaster subgroup, even informative for the D. melanogaster lineage. Contemporary crossover rates in D. melanogaster, on the other hand, do not recapitulate ancestral crossovers landscapes. As a result, the temporal stability of crossover landscapes observed in D. yakuba makes this species an ideal system for applying population genetic models of selection and linkage, given that these models assume temporal constancy in linkage effects. Our studies emphasize the importance of generating multiple high-resolution crossover rate maps within a coherent phylogenetic context to broaden our understanding of crossover control during meiosis and to improve studies on the evolutionary consequences of variable crossover rates across genomes and time.     

Repeat instability at human minisatellites arising from meiotic recombination.

Little is known about the role of meiotic recombination processes such as unequal crossover in driving instability at tandem repeat DNA. Methods have therefore been developed to detect meiotic crossovers within two different GC-rich minisatellite repeat arrays in humans, both in families and in sperm DNA. Both loci normally mutate in the germline by complex conversion-like transfer of repeats between alleles. Analysis shows that inter-allelic unequal crossovers also occur at both loci, although at low frequency, to yield simple recombinant repeat arrays with exchange of flanking markers. Equal crossovers between aligned alleles, resulting in recombinant alleles but without change in repeat copy number, also occur in sperm at a similar frequency to unequal crossovers. Both crossover and conversion show polarity in the repeat array and are co-suppressed in an allele showing unusual germline stability. This provides evidence that minisatellite conversion and crossover arise by a common mechanism, perhaps by alternative processing of a meiotic recombination initiation complex, and implies that minisatellite instability is a by-product of meiotic recombination in repeat DNA. While minisatellite recombination is infrequent, crossover rates indicate that the unstable end of a human minisatellite can act as a recombination warm-spot, even between sequence-heterologous alleles.     

Meiotic crossing-over in nondisjoined chromosomes of children with trisomy 21 and a congenital heart defect.

We have used DNA polymorphisms to study meiotic crossovers of chromosome 21q in 27 nuclear families. Each family had a child with Down syndrome and a congenital heart defect. Twenty DNA polymorphisms on chromosome 21 were used to determine parental and meiotic origin of nondisjunction and to identify crossovers. Twenty-four cases were of maternal origin, and three were of paternal origin. Twenty-two unequivocal crossover events were identified. Sixteen crossovers were observed in 22 chromosome pairs nondisjoining at the second meiotic division. Fifty percent of crossover events in MI nondisjunction are detectable by molecular genetic means. Thus, the results suggest that, in this sample, each nondisjoined chromosome 21 pair has been involved in at least one crossover event.     

The road to crossovers: plants have their say

Crossovers involve the reciprocal exchange of large fragments of genetic material between homologous chromosomes during meiosis. In this way, crossovers are the basis of genetics. Remarkably, the number and distribution of crossovers on chromosomes are closely controlled. Data from various model organisms (notably Saccharomyces cerevisiae) show that the distribution of crossovers results from a series of tightly regulated events involving the formation and repair of double-strand breaks and interference. Recent advances in genetic and cytological tools, particularly for studying Arabidopsis thaliana, have enabled crossover control in plants to be studied in more detail. In this article, we discuss the contribution of plant studies to meiosis research, particularly to our understanding of crossover control and interference, and we evaluate models of interference.