Genetic Evidence for Preferential Strand Transfer during Meiotic Recombination in Yeast

During meiotic recombination in the yeast Saccharomyces cerevisiae, heteroduplexes are formed as an intermediate in the exchange process. In the formation of an asymmetric heteroduplex, one chromosome acts as a donor of a single DNA strand and the other acts as a recipient. We present genetic evidence that the nontranscribed strand is donated more frequently than the transcribed strand in spores that have an unrepaired mismatch at the HIS4 locus.     

Recombination can evolve in large finite populations given selection on sufficient loci

It is well known that an allele causing increased recombination is expected to proliferate as a result of genetic drift in a finite population undergoing selection, without requiring other mechanisms. This is supported by recent simulations apparently demonstrating that, in small populations, drift is more important than epistasis in increasing recombination, with this effect disappearing in larger finite populations. However, recent experimental evidence finds a greater advantage for recombination in larger populations. These results are reconciled by demonstrating through simulation without epistasis that for m loci recombination has an appreciable selective advantage over a range of population sizes (am, bm). bm increases steadily with m while am remains fairly static. Thus, however large the finite population, if selection acts on sufficiently many loci, an allele that increases recombination is selected for. We show that as selection acts on our finite population, recombination increases the variance in expected log fitness, causing indirect selection on a recombination-modifying locus. This effect is enhanced in those populations with more loci because the variance in phenotypic fitnesses in relation to the possible range will be smaller. Thus fixation of a particular haplotype is less likely to occur, increasing the advantage of recombination.     

The Evolutionary Advantage of Recombination. II. Individual Selection for Recombination

Based on the Fisher-Muller theory of the evolution of recombination, an argument can be constructed predicting that a recessive allele favoring recombination will be favored, if there are either favorable or deleterious mutants occurring at other loci. In this case there is no clear distinction between individual and group selection. Computer simulation of populations segregating for recessive or dominant recombination alleles showed selection favoring recombination, except in the case of a dominant recombination allele with deleterious background mutants. The relationship of this work to parallel investigations by Williams and by Strobeck, Maynard Smith, and Charlesworth is explored. All seem to rely on the same phenomenon. There seems no reason to assume that the evolution of recombination must have occurred by group selection.     

Recombination and meiosis.

Although exchanges between sister chromatids are common in mitotic cells, those involving homologous chromosomes are rare. Since recombination between homologues is one of the functions of meiosis, it follows that one aspect of the differentiation of the meiocyte involves the synthesis of proteins or enzymes which facilitate synapsis and exchange. Mutants are known which seem to have constitutive levels of mitotic recombination between homologues, and these may be defective in the mechanism which normally represses mitotic recombination. It has been proposed that one component of the synaptonemal complex (s.c.) is a filamentous pairing protein with DNA binding sites which are base sequence specific. Synapsis occurs because the distribution of these sequences is the same in homologues. When only non-homologous chromosomes are present, as in haploid meiosis, only weak pairing can occur, since the base sequences are largely out of register. Although certain features of recombination at the molecular level are known, none of the models so far proposed suggest an explanation for interference between crossovers. It is suggested that interference may depend on the presence of a limited amount of another DNA binding protein which is specifically located within the s.c. A crossover between naked DNA molecules is initially a weak structure, which must be later converted into a visible and mechanically strong chiasma. It is assumed that this stabilization of a crossover is achieved by the DNA binding protein, which can diffuse freely within the s.c. and bind cooperatively to any recombinant DNA molecules within it. Depletion of the binding protein within the vicinity of a crossover makes it unlikely that the second crossover can be formed nearby.     

Me14, a Yeast Gene Required for Meiotic Recombination

Mutants at the MEI4 locus were detected in a search for mutants defective in meiotic gene conversion. mei4 mutants exhibit decreased sporulation and produce inviable spores. The spore inviability phenotype is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The MEI4 gene has been cloned from a yeast genomic library by complementation of the recombination defect and has been mapped to chromosome V near gln3. Strains carrying a deletion/insertion mutation of the MEI4 gene display no meiotically induced gene conversion but normal mitotic conversion frequencies. Both meiotic interchromosomal and intrachromosomal crossing over are completely abolished in mei4 strains. The mei4 mutation is able to rescue the spore-inviability phenotype of spo13 and 52 strains (i.e., mei4 spo13 rad52 mutants produce viable spores), indicating that MEI4 acts before RAD52 in the meiotic recombination pathway.     

The Effect of Single Recombination Events on Coalescent Tree Height and Shape

The coalescent with recombination is a fundamental model to describe the genealogical history of DNA sequence samples from recombining organisms. Considering recombination as a process which acts along genomes and which creates sequence segments with shared ancestry, we study the influence of single recombination events upon tree characteristics of the coalescent. We focus on properties such as tree height and tree balance and quantify analytically the changes in these quantities incurred by recombination in terms of probability distributions. We find that changes in tree topology are often relatively mild under conditions of neutral evolution, while changes in tree height are on average quite large. Our results add to a quantitative understanding of the spatial coalescent and provide the neutral reference to which the impact by other evolutionary scenarios, for instance tree distortion by selective sweeps, can be compared.     

Coincident Recombination during Mitosis in Saccharomyces: Distance-Dependent and -Independent Components

In mitosis, coincident recombination events between widely separated markers occur more frequently than expected for two independent acts. Several different mechanisms have been proposed to account for this phenomenon. It has been argued that coincident recombination could be due to either an extensive region of heteroduplex DNA or some other distance-dependent mechanism. Alternately, it has been suggested that at least some is due to subpopulations of cells which undergo recombination at very high frequencies. The purpose of these experiments is to evaluate the possible contribution of distance-dependent and distance-independent components. By comparing the coincident recombination frequencies for markers on the same homolog as well as pairs of unlinked sites, we show that there is a strong distance-dependent component for at least 8.8-35-kbp, depending on the type of recombination event (conversion or intrachromosomal exchange). For larger distances separating sites, a distance-independent mechanism(s) results in higher than expected frequencies.     

Localized Conversion in STREPTOCOCCUS PNEUMONIAE Recombination: Heteroduplex Preference

In pneumococcal transformation the frequency of recombinants between point mutations is generally proportional to distance. We have recently described an aberrant marker in the amiA locus that appeared to enhance recombination frequency when crossed with any other allele of this gene. The hyperrecombination that we have observed in two-point crosses could be explained by two hypotheses: the aberrant marker induces frequent crossovers in its vicinity or the mutant is converted to wild type. In this report we present evidence showing that, in suitable three-point crosses, this hyperrecombination does not modify the recombination frequency between outside markers, suggesting that a conversion occurs at the site of this mutation. To estimate the length over which this event occurs, we isolated very closely linked markers and used them in two-point crosses. It appears that the conversion system removes only a few base pairs (from three to 27) around the aberrant marker. This conversion process is quite different from the mismatch-repair system controlled by hex genes in pneumococcus, which involves several thousand base pairs. Moreover, we have constructed artificial heteroduplexes using separated DNA strands. It appears that only one of the two heteroduplexes is specifically converted. The conversion system acts upon 5'..ATTAAT..3'/3'..TAAGTA..5'. A possible role of the palindrome resulting from the mutation is discussed.     

Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation.     

A PHF8 Homolog in C. elegans Promotes DNA Repair via Homologous Recombination

PHF8 is a JmjC domain-containing histone demethylase, defects in which are associated with X-linked mental retardation. In this study, we examined the roles of two PHF8 homologs, JMJD-1.1 and JMJD-1.2, in the model organism C. elegans in response to DNA damage. A deletion mutation in either of the genes led to hypersensitivity to interstrand DNA crosslinks (ICLs), while only mutation of jmjd-1.1 resulted in hypersensitivity to double-strand DNA breaks (DSBs). In response to ICLs, JMJD-1.1 did not affect the focus formation of FCD-2, a homolog of FANCD2, a key protein in the Fanconi anemia pathway. However, the dynamic behavior of RPA-1 and RAD-51 was affected by the mutation: the accumulations of both proteins at ICLs appeared normal, but their subsequent disappearance was retarded, suggesting that later steps of homologous recombination were defective. Similar changes in the dynamic behavior of RPA-1 and RAD-51 were seen in response to DSBs, supporting a role of JMJD-1.1 in homologous recombination. Such a role was also supported by our finding that the hypersensitivity of jmjd-1.1 worms to ICLs was rescued by knockdown of lig-4, a homolog of Ligase 4 active in nonhomologous end-joining. The hypersensitivity of jmjd-1.1 worms to ICLs was increased by rad-54 knockdown, suggesting that JMJD-1.1 acts in parallel with RAD-54 in modulating chromatin structure. Indeed, the level of histone H3 Lys9 tri-methylation, a marker of heterochromatin, was higher in jmjd-1.1 cells than in wild-type cells. We conclude that the histone demethylase JMJD-1.1 influences homologous recombination either by relaxing heterochromatin structure or by indirectly regulating the expression of multiple genes affecting DNA repair.     

Recombination values and their errors.

Two four-point testcrosses comprising 87,000 tomato plants were grown and the data collected from 28 subgroups. Each subgroup consisted of 2,000 or 5,000 plants and should give a valid estimate of the three recombination values. The 28 values for each interval give more outlyers (23% are outside the 95% limits set by the standard deviation calculated by the binomial formula square root of p q/n) than would be expected by chance. If each subgroup was regarded as the control and the other groups tested against this, then 42% of the time the two subgroups would be significantly different. It is suggested that there are many cases in the literature where this comparison has been made and the significant difference wrongly ascribed to treatment. While the causes of these changes in recombination value are unknown and therefore uncontrollable, they must be anticipated in all such studies. Control and treatment must be replicated enough that chance extreme values will not be attributed to treatment.     

Recombination of uracil-containing lambda bacteriophages.

Controlled incorporation of uracil into the deoxyribonucleic acid (DNA) of lambda bacteriophages was achieved by growth on dut ung thy mutants of Escherichia coli. The frequency of substitution of uracil for thymine, estimated by alkaline sucrose sedimentation of phage DNA treated in vitro with uracil DNA glycosylase, ranged from 0.17 to 1.9%. The corresponding ratio between the plating efficiencies on wild-type (Ung+) and glycosylase-deficient (Ung-) bacteria ranged from 0.70 to 0.05. If a single-hit dependence of plating efficiency on uracil content is assumed, the probability that any given uracil residue is lethal is approximately 1% (about one-fifth the probability for a pyrimidine dimer). The effect of uracil on recombination was studied in experiments with lambda tandem duplication phages (ethylenediaminetetraacetic acid [EDTA] sensitive), which are converted to single-copy phages (EDTA resistant) by general recombination. For repressed infections (of homoimmune lysogens), recombination was measured by a two-stage assay (DNA extraction, transfection of spheroplasts, and EDTA treatment). The frequencies observed for uracil-containing phages (2 to 4%) were 5 to 10 times higher than control values. However, comparisons with ultraviolet irradiated phages indicated that uracil residues promoted recombination less than 1/100 as efficiently as ultraviolet-induced lesions. Recombination of uracil-containing phages during repressed infections was negligible in recA and partially reduced in recB bacteria. Recombination was very low in ung cells, suggesting that excision repair was responsible for the stimulation. Interestingly, uracil-stimulated recombination was elevated about twofold in xth bacteria.     

Genetic Recombination in Coprinus. IV. a Kinetic Study of the Temperature Effect on Recombination Frequency

At the restrictive conditions (35 degrees under continuous light) Coprinus lagopus is unable to initiate premeiotic S phase which takes place normally within 8-10 h of karyogamy. A shift-up to the restrictive conditions causes an arrest of the basidiocarps at this critical stage. A prolonged arrest causes a reversal to mitosis (Lu 1974b). Incubation of basidiocarps at the restrictive conditions before this critical stage causes no increase in recombination frequency (R.F.) in the loci studied. An arrest of 4 h at the critical stage still causes no R.F. increase, but 12-13 h and 18-19 h arrests cause increases of 50% and 90% over the controls, respectively. Thus R.F. can be increased even before the cells are fully committed to meiosis.-A 3-h heat treatment at the beginning of S phase (or 8 h before karyogamy) also causes some (30%) increase in R.F. while the same treatment at late S phase (or 3 h before karyogamy) causes a substantial (164%) increase in R.F. over the controls. A 3-h heat treatment before S phase causes no increase in R.F.-Pachytene is also responsive to temperature treatments (Lu 1969). The maximum R.f. increase is 100% by heat and 220% by cold treatment. The shortest time that can cause the maximum increase in recombination by high temperature is 3 h and that by cold treatment is 7 h. These durations are correlated with the length of the pachytene stage under the treatment conditions. The kinetic data show that the increase in R.F. caused by high and low temperatures follows two-hit kinetics and their rate of increase is almost identical. The higher increase in R.F. by low temperature can be attributed to the increased duration of pachytene and therefore R.F. is a function of time. The longer the homologous chromosomes are held together, the higher the recombination frequency.     

Recombination mediator and Rad51 targeting activities of a human BRCA2 polypeptide

BRCA2 likely exerts its tumor suppressor function by enhancing the efficiency of the homology-directed repair of injured chromosomes. To help define the DNA repair role of BRCA2, we expressed and purified a polypeptide, BRC3/4-DBD, that harbors its BRC3 and BRC4 repeats and DNA binding domain. BRC3/4-DBD interacted with hRad51 and bound DNA with a distinct preference for single-stranded (ss) DNA. Importantly we demonstrated by biochemical means and electron microscopy that BRC3/4-DBD nucleates hRad51 onto ssDNA and acts as a recombination mediator in enabling hRad51 to utilize replication protein A-coated ssDNA as recombination substrate. These functions of BRC3/4-DBD required both the BRC repeats and the BRCA2 DNA binding domain. The results thus clarify the role of BRCA2 in Rad51-dependent DNA recombination and repair, and the experimental strategies described herein should be valuable for systematically deciphering this BRCA2 function.     

The Rec102 Mutant of Yeast Is Defective in Meiotic Recombination and Chromosome Synapsis

A mutation at the REC102 locus was identified in a screen for yeast mutants that produce inviable spores. rec102 spore lethality is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The rec102 mutation completely eliminates meiotically induced gene conversion and crossing over but has no effect on mitotic recombination frequencies. Cytological studies indicate that the rec102 mutant makes axial elements (precursors to the synaptonemal complex), but homologous chromosomes fail to synapse. In addition, meiotic chromosome segregation is significantly delayed in rec102 strains. Studies of double and triple mutants indicate that the REC102 protein acts before the RAD52 gene product in the meiotic recombination pathway. The REC102 gene was cloned based on complementation of the mutant defect and the gene was mapped to chromosome XII between CDC25 and STE11.     

Rad51 Paralog Complexes BCDX2 and CX3 Act at Different Stages in the BRCA1-BRCA2-Dependent Homologous Recombination Pathway

The Rad51 paralogs are required for homologous recombination (HR) and the maintenance of genomic stability. The molecular mechanisms by which the five vertebrate Rad51 paralogs regulate HR and genomic integrity remain unclear. The Rad51 paralogs associate with one another in two distinct complexes: Rad51B-Rad51C-Rad51D-XRCC2 (BCDX2) and Rad51C-XRCC3 (CX3). We find that the BCDX2 and CX3 complexes act at different stages of the HR pathway. In response to DNA damage, the BCDX2 complex acts downstream of BRCA2 recruitment but upstream of Rad51 recruitment. In contrast, the CX3 complex acts downstream of Rad51 recruitment but still has a marked impact on the measured frequency of homologous recombination. Both complexes are epistatic with BRCA2 and synthetically lethal with Rad52. We conclude that human Rad51 paralogs facilitate BRCA2-Rad51-dependent homologous recombination at different stages in the pathway and function independently of Rad52.     

Genes in Neurospora That Suppress Recombination When They Are Heterozygous

Genes that suppress recombination when heterozygous have been found distributed as a polymorphism in wild and laboratory populations of Neurospora crassa. Three alleles, ss^E, ss^S and ss^C, are associated, respectively, with the three wild types Emerson, St. Lawrence 74A and Costa Rica A. It is proposed that ss (synaptic sequence) genes modulate recombination by determining the pairing closeness of DNA duplexes in the vicinity of the nit-2 locus. When heterozygous, ss suppresses recombination 2- to 20-fold within the nit-2 locus, which it adjoins, but crossing over in intervals flanking nit-2 is not affected. The magnitude of suppression depends upon the ss alleles involved, and ss acts multiplicatively with rec-1; together, these genes modulate recombination within the nit-2 locus over a range exceeding 100-fold. The ss effect is not attributable to gross chromosomal rearrangement, but could be due to small inversions or insertions, such as transposable elements.     

hpr1Δ Affects Ribosomal DNA Recombination and Cell Life Span in Saccharomyces cerevisiae

Multiple genetic pathways have been shown to regulate life span and aging in the yeast Saccharomyces cerevisiae. Here we show that loss of a component of the RNA polymerase II complex, Hpr1p, results in a decreased life span. Although hpr1Delta mutants have an increased rate of recombination within the ribosomal DNA (rDNA) array, this is not accompanied by an increase in extrachromosomal rDNA circles (ERCs). Analyses of mutants that affect replication of the rDNA array and suppressors that reverse the phenotypes of the hpr1Delta mutant show that the reduced life span is associated with increased genomic instability but not with increased ERC formation. The hpr1Delta mutant acts in a pathway distinct from previously described mutants that reduce life span.