The impact of sequence divergence and DNA mismatch repair on homeologous recombination in Arabidopsis

We examined the effects of substrate divergence and DNA mismatch repair (MMR) on recombination in Arabidopsis thaliana. Relative to the frequency observed in plants with a homologous construct (0% divergence), recombination was decreased 4.1-, 9.6-, 11.7- or 20.3-fold, respectively, in lines with constructs containing 0.5%, 2%, 4% or 9% divergence between the recombination substrates. To evaluate the contribution of the MMR system in this decrease, 12 independent reporter lines (two or three lines per reporter construct) were crossed to an AtMSH2 T-DNA insertional mutant. We examined the recombination frequency in progeny homozygous for a reporter T-DNA and homozygous either for the wild type or the mutant allele of AtMSH2. The loss of MMR activity led to a two- to ninefold increase in homeologous recombination and the size of the increase did not seem to correlate with the amount of divergence. Inversely, complementation of the insertional mutant with a wild-type cDNA of AtMSH2 reduced recombination. Our results demonstrate clearly that sequence divergence can dramatically reduce the recombination frequency in plants and that the MMR system plays a part in this decrease.     

Asymmetric random walk in a reaction intermediate of homologous recombination

At an intermediate step of the homologous recombination between two double-stranded DNA molecules, a point (often called Holliday structure) connecting two strands coming from two recombining partners migrates along the homologous region. Assuming random walk of a connecting point, we previously explained the dependence of recombination frequency on the homology length observed in vivo. In this model, the random walk was assumed to be symmetric in that the forward transition rate equals the backward one. According to observations in vitro, however, catalysed migration appears unidirectional. Taking into account possible asymmetry, we thus reformulate our random walk model to reexamine the observations in vivo. We also derive some theoretical results to analyse dynamic processes observed in vitro.     

The minimum amount of homology required for homologous recombination in mammalian cells.

Although DNA sequence homology is believed to be a prerequisite for homologous recombination events in procaryotes and eucaryotes, no systematic study has been done on the minimum amount of homology required for homologous recombination in mammalian cells. We have used simian virus 40-pBR322 hybrid plasmids constructed in vitro as substrates to quantitate intramolecular homologous recombination in cultured monkey cells. Excision of wild-type simian virus 40 DNA by homologous recombination was scored by the viral plaque assay. Using a series of plasmids containing 0 to 243 base pairs of homology, we have shown that the recombination frequency decreases as the homology is reduced, with the sharpest drop in recombination frequency occurring when the homology was reduced from 214 to 163 base pairs. However, low recombination frequencies were also observed with as little as 14 base pairs of homology.     

Cellular response to horizontally transferred DNA in Escherichia coli is tuned by DNA repair systems

We studied how DNA divergence between recombining DNAs and the mismatch repair system modulate the SOS response in Escherichia coli. The observed positive log-linear correlation between SOS induction and DNA divergence, and the negative correlation between SOS induction and frequency of recombination, suggest that the level of SOS induction precisely reflects the difficulty of RecA protein to initiate a productive strand exchange process. Our results suggest that the mismatch repair system could contribute to this SOS induction more by affecting the RecA-catalyzed homology search than by acting on mismatched recombination intermediates. The propensity of the recombination machinery to promote recombination between the blocks of sequences with the highest identity results in the increasing ratios of merodiploids (partial diploids) over genuine recombinants (homologous replacements) with increasing DNA divergence. We discuss the role of molecular mechanisms involved in the control of the recombination between diverged DNA sequences in the maintenance of genomic stability and genome evolution.     

Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology

Summary To determine the minimal DNA sequence homology required for recombination in Bacillus subtilis, we developed a system capable of distinguishing between homologous and illegitimate recombination events during plasmid integration into the chromosome. In this system the recombination frequencies were measured between is pE194 derivatives carrying segments of the chromosomal -gluconase gene (bglS) of various lengths and the bacterial chromosome, using selection for erythromycin resistance at the non-permissive temperature. Homologous recombination events, resulting in disruption of the bglS gene, were easily detected by a colorimetric assay for -gluconase activity. A linear dependence of recombination frequency on homology length was observed over an interval of 77 bp. It was found that approximately 70 bp of homology is required for detectable homologous recombination. Homologous recombination was not detected when only 25 by of homology between plasmid and chromosome were provided. The data indicate that homology requirements for recombination in B. subtilis differ from those in Escherichia coli.     

Parameters controlling the gene-targeting frequency at the Sphingomonas species rrn site and expression of the methyl parathion hydrolase gene

AIMS: To investigate the key parameters controlling the exogenous methyl parathion hydrolase (MPH) gene mpd-targeting frequency at the ribosomal RNA operon (rrn) site of Sphingomonas species which has a wide range of biotechnological applications. METHODS AND RESULTS: Targeting vectors with different homology lengths and recipient target DNA with different homology identities were used to investigate the parameters controlling the targeting frequency at the Sphingomonas species rrn site. Targeting frequency decreased with the reduction of homology length, and the minimal size for normal homologous recombination was >100 bp. Homologous recombination could succeed even if there were 3-4% mismatches; however, targeting frequency decreased with increasing sequence divergence. The Red recombination system could increase the targeting frequency to some extent. Targeting of the mpd gene to the rrn site did not affect cell viability and resulted in an increase of MPH-specific activity in recombinants. CONCLUSIONS: Targeting frequency was affected by homology length, identity and the Red recombination system. The rrn site is a good target site for the expression of exogenous genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is useful as a foundation for a better understanding of recombination events involving homologous sequences and for the improved manipulation of Sphingomonas genes in biotechnological applications.     

Avoidance of inter-repeat recombination by sequence divergence and a mechanism of neutral evolution

Eucaryotic genomes are loaded with diverse repeated sequences and are therefore threatened by rearrangements via inter-repeat crossovers and by gene-inactivating conversions between genes and their inactive pseudogenes. Such repeated DNA sequences are usually diverged and polymorphic. Sequence divergence by well-spread point mutations is a potent inhibitor of homologous recombination due to the loss of recombination initiation sites and to the editing of recombinational intermediates by the mismatch repair system. Evidence is reviewed suggesting that a germ line process can identify duplicated sequences by homologous pairing, modify them by methylation and mutate by C----T transitions. Since this process requires a minimum contiguous homology that is larger than the average exon size, it is proposed that fragmentation by intron inserts protects the coding sequences from inactivation by homologous interactions with their pseudogene sequences.     

Dependence of Frequency of Homologous Recombination on the Homology Length

The frequency of homologous recombination is believed to be a linear function of the length (N bp) of homology between DNAs. Here, the N intercept is believed to be determined by a threshold length below which some physical constraint is effective. In the mammalian gene targeting systems, however, the frequency depends more steeply than linearly on the homology length. To explain both the linear dependence and the steeper dependence, we propose a model where the branch point of a reaction intermediate is assumed to ``walk randomly' along the homologous region until it is processed. The intermediate is assumed to be destroyed if the branch point ever reaches either end of the homology. In this model, the length dependence is governed by a parameter, h, which is defined as efficiency of processing of the intermediate and reflects unlikelihood of the destruction at either end of the homology. We find that the frequency is proportional to N(3) for smaller N and is a linear function of N for larger N. Where the shift from the N(3) dependence to the linear dependence takes place is determined by the parameter h. The range of N showing the N(3) dependence becomes narrower as h becomes larger. The dependence steeper than linear dependence, which is observed not only in the mammalian gene targeting system but also in bacteriophage T4, Escherichia coli and yeast systems, agrees well with the predicted N(3) dependence. The N intercept is determined not by physical (or structural) constraints but only by the parameter h in this model.     

Repair of double-strand breaks by homologous recombination in mismatch repair-defective mammalian cells

Chromosomal double-strand breaks (DSBs) stimulate homologous recombination by several orders of magnitude in mammalian cells, including murine embryonic stem (ES) cells, but the efficiency of recombination decreases as the heterology between the repair substrates increases (B. Elliott, C. Richardson, J. Winderbaum, J. A. Nickoloff, and M. Jasin, Mol. Cell. Biol. 18:93-101, 1998). We have now examined homologous recombination in mismatch repair (MMR)-defective ES cells to investigate both the frequency of recombination and the outcome of events. Using cells with a targeted mutation in the msh2 gene, we found that the barrier to recombination between diverged substrates is relaxed for both gene targeting and intrachromosomal recombination. Thus, substrates with 1.5% divergence are 10-fold more likely to undergo DSB-promoted recombination in Msh2(-/-) cells than in wild-type cells. Although mutant cells can repair DSBs efficiently, examination of gene conversion tracts in recombinants demonstrates that they cannot efficiently correct mismatched heteroduplex DNA (hDNA) that is formed adjacent to the DSB. As a result, >20-fold more of the recombinants derived from mutant cells have uncorrected tracts compared with recombinants from wild-type cells. The results indicate that gene conversion repair of DSBs in mammalian cells frequently involves mismatch correction of hDNA rather than double-strand gap formation. In cells with MMR defects, therefore, aberrant recombinational repair may be an additional mechanism that contributes to genomic instability and possibly tumorigenesis.     

Homologous Recombination in ESCHERICHIA COLI: Dependence on Substrate Length and Homology

We studied the in vivo recombination between homologous DNA sequences cloned in phage lambda and a pBR322-derived plasmid by assaying for the formation of phage-plasmid cointegrates by a single (or an odd number of) reciprocal exchange. (1) Recombination proceeds by the RecBC pathway in wild-type cells and by low levels of a RecF-dependent pathway in recBC- cells. The RecE pathway appears not to generate phage-plasmid cointegrates. (2) Recombination is linearly dependent on the length of the homologous sequences. In both RecBC and RecF-dependent pathways there is a minimal length, called the minimal efficient processing segment (MEPS), below which recombination becomes inefficient. The length of MEPS is between 23-27 base pairs (bp) and between 44-90 bp for the RecBC- and RecF-dependent pathways, respectively. A model, based on overlapping MEPS, of the correlation of genetic length with physical length is presented. The bases for the different MEPS length of the two pathways are discussed in relationship to the enzymes specific to each pathway. (3) The RecBC and the RecF-dependent pathways are each very sensitive to substrate homology. In wild-type E. coli, reduction of homology from 100% to 90% decreases recombinant frequency over 40-fold. The homology dependence of the RecBC and RecF-dependent pathways are similar. This suggests that a component common to both, probably recA, is responsible for the recognition of homology.     

High frequency induction of mitotic recombination by ionizing radiation in Mlh1 null mouse cells

Mitotic recombination in somatic cells involves crossover events between homologous autosomal chromosomes. This process can convert a cell with a heterozygous deficiency to one with a homozygous deficiency if a mutant allele is present on one of the two homologous autosomes. Thus mitotic recombination often represents the second mutational step in tumor suppressor gene inactivation. In this study we examined the frequency and spectrum of ionizing radiation (IR)-induced autosomal mutations affecting Aprt expression in a mouse kidney cell line null for the Mlh1 mismatch repair (MMR) gene. The mutant frequency results demonstrated high frequency induction of mutations by IR exposure and the spectral analysis revealed that most of this response was due to the induction of mitotic recombinational events. High frequency induction of mitotic recombination was not observed in a DNA repair-proficient cell line or in a cell line with an MMR-independent mutator phenotype. These results demonstrate that IR exposure can initiate a process leading to mitotic recombinational events and that MMR function suppresses these events from occurring.     

Induction of recombination between homologous and diverged DNAs by double-strand gaps and breaks and role of mismatch repair.

Sequence homology is expected to influence recombination. To further understand mechanisms of recombination and the impact of reduced homology, we examined recombination during transformation between plasmid-borne DNA flanking a double-strand break (DSB) or gap and its chromosomal homolog. Previous reports have concentrated on spontaneous recombination or initiation by undefined lesions. Sequence divergence of approximately 16% reduced transformation frequencies by at least 10-fold. Gene conversion patterns associated with double-strand gap repair of episomal plasmids or with plasmid integration were analyzed by restriction endonuclease mapping and DNA sequencing. For episomal plasmids carrying homeologous DNA, at least one input end was always preserved beyond 10 bp, whereas for plasmids carrying homologous DNA, both input ends were converted beyond 80 bp in 60% of the transformants. The system allowed the recovery of transformants carrying mixtures of recombinant molecules that might arise if heteroduplex DNA--a presumed recombination intermediate--escapes mismatch repair. Gene conversion involving homologous DNAs frequently involved DNA mismatch repair, directed to a broken strand. A mutation in the PMS1 mismatch repair gene significantly increased the fraction of transformants carrying a mixture of plasmids for homologous DNAs, indicating that PMS1 can participate in DSB-initiated recombination. Since nearly all transformants involving homeologous DNAs carried a single recombinant plasmid in both Pms+ and Pms- strains, stable heteroduplex DNA appears less likely than for homologous DNAs. Regardless of homology, gene conversion does not appear to occur by nucleolytic expansion of a DSB to a gap prior to recombination. The results with homeologous DNAs are consistent with a recombinational repair model that we propose does not require the formation of stable heteroduplex DNA but instead involves other homology-dependent interactions that allow recombination-dependent DNA synthesis.     

Formation and repair of heteroduplex DNA on both sides of the double-strand break during mammalian gene targeting

In this study, we examined homologous recombination in mammalian cells using a gene targeting assay in which the introduction of a double-strand-break (DSB) in the vector-borne region of homology to the chromosome resulted in targeted vector integration. The vector-borne DSB was flanked with small palindromic insertions that, when encompassed within heteroduplex DNA (hDNA) formed during targeted vector integration, were capable of avoiding the activity of the mismatch repair (MMR) system. When used in conjunction with an isolation procedure in which the product(s) of each targeted vector integration event were retained for molecular analysis, information about recombination mechanisms was obtained. The examination of marker segregation patterns in independent recombinants revealed the following, (i) hDNA tracts could form simultaneously on each side of the DSB and in both participating homologous regions. Clonal analysis of sectored recombinants revealed that, in the homologous repeats generated by the recombination event, vector-borne palindrome and chromosomal markers were linked in the expected way in each strand of the hDNA intermediate, (ii) hDNA tracts were subject to MMR processing that occurred on opposite sides of the DSB, and (iii) in the majority of recombinants, the vector-borne marker was replaced with the corresponding marker from the chromosome. Bidirectional hDNA formation and MMR processing of both sides of the DSB are consistent with the double-strand-break repair (DSBR) model of recombination.     

The role of the mismatch repair machinery in regulating mitotic and meiotic recombination between diverged sequences in yeast

Nonidentical recombination substrates recombine less efficiently than do identical substrates in yeast, and much of this inhibition can be attributed to action of the mismatch repair (MMR) machinery. In this study an intron-based inverted repeat assay system has been used to directly compare the rates of mitotic and meiotic recombination between pairs of 350-bp substrates varying from 82% to 100% in sequence identity. The recombination rate data indicate that sequence divergence impacts mitotic and meiotic recombination similarly, although subtle differences are evident. In addition to assessing recombination rates as a function of sequence divergence, the endpoints of mitotic and meiotic recombination events involving 94%-identical substrates were determined by DNA sequencing. The endpoint analysis indicates that the extent of meiotic heteroduplex DNA formed in a MMR-defective strain is 65% longer than that formed in a wild-type strain. These data are consistent with a model in which the MMR machinery interferes with the formation and/or extension of heteroduplex intermediates during recombination.     

Type I restriction enzyme with RecA protein promotes illegitimate recombination

Illegitimate (non-homologous) recombination requires little or no sequence homology between recombining DNAs and has been regarded as being a process distinct from homologous recombination, which requires a long stretch of homology between recombining DNAs. However, we have found a type of illegitimate recombination that requires an interaction between long homologous DNA sequences. It was detected when a plasmid that carried 2-kb-long inverted repeats was subjected to type I (EcoKI) restriction in vivo within a special mutant strain of Escherichia coli. In the present work, we analyzed genetic requirements for this type of illegitimate recombination in well-defined genetic backgrounds. Our analysis demonstrated dependence on RecA function and on the presence of two EcoKI sites on the substrate DNA. These results are in harmony with a model in which EcoKI restriction enzyme attacks an intermediate of homologous recombination to divert it to illegitimate recombination.     

Homology Search and Choice of Homologous Partner during Mitotic Recombination

Homologous recombination is an important DNA repair mechanism in vegetative cells. During the repair of double-strand breaks, genetic information is transferred between the interacting DNA sequences (gene conversion). This event is often accompanied by a reciprocal exchange between the homologous molecules, resulting in crossing over. The repair of DNA damage by homologous recombination with repeated sequences dispersed throughout the genome might result in chromosomal aberrations or in the inactivation of genes. It is therefore important to understand how the suitable homologous partner for recombination is chosen. We have developed a system in the yeast Saccharomyces cerevisiae that can monitor the fate of a chromosomal double-strand break without the need to select for recombinants. The broken chromosome is efficiently repaired by recombination with one of two potential partners located elsewhere in the genome. One of the partners has homology to the broken ends of the chromosome, whereas the other is homologous to sequences distant from the break. Surprisingly, a large proportion of the repair is carried out by recombination involving the sequences distant from the broken ends. This repair is very efficient, despite the fact that it requires the processing of a large chromosomal region flanking the break. Our results imply that the homology search involves extensive regions of the broken chromosome and is not carried out exclusively by sequences adjacent to the double-strand break. We show that the mechanism that governs the choice of homologous partners is affected by the length and sequence divergence of the interacting partners, as well as by mutations in the mismatch repair genes. We present a model to explain how the suitable homologous partner is chosen during recombinational repair. The model provides a mechanism that may guard the integrity of the genome by preventing recombination between dispersed repeated sequences.     

Barriers to genetic exchange between bacterial species: Streptococcus pneumoniae transformation

Interspecies genetic exchange is an important evolutionary mechanism in bacteria. It allows rapid acquisition of novel functions by transmission of adaptive genes between related species. However, the frequency of homologous recombination between bacterial species decreases sharply with the extent of DNA sequence divergence between the donor and the recipient. In Bacillus and Escherichia, this sexual isolation has been shown to be an exponential function of sequence divergence. Here we demonstrate that sexual isolation in transformation between Streptococcus pneumoniae recipient strains and donor DNA from related strains and species follows the described exponential relationship. We show that the Hex mismatch repair system poses a significant barrier to recombination over the entire range of sequence divergence (0.6 to 27%) investigated. Although mismatch repair becomes partially saturated, it is responsible for 34% of the observed sexual isolation. This is greater than the role of mismatch repair in Bacillus but less than that in Escherichia. The remaining non-Hex-mediated barrier to recombination can be provided by a variety of mechanisms. We discuss the possible additional mechanisms of sexual isolation, in view of earlier findings from Bacillus, Escherichia, and Streptococcus.     

Interference of DNA sequence divergence with precise recombinational DNA repair in mammalian cells.

Studies done in prokaryotes and eukaryotes have indicated that DNA sequence divergence decreases the frequency of homologous recombination. To determine which step(s) of homologous recombination is sensitive to DNA sequence divergence in mammalian cells we have used an assay that does not rely on the recovery of functional products. The assay is based on the acquisition by homologous recombination of endogenous LINE-1 sequences by exogenous LINE-1 sequences. In parallel experiments, we introduced into mouse cells two gapped exogenous LINE-1 sequences, one from the mouse, L1Md-A2, and the other from the rat, L1Rn-3. Although L1Rn-3 is on average less than 85% homologous to the LINE-1 elements of the mouse, the frequency of homologous recombination with endogenous LINE-1 elements obtained with L1Rn-3 was the same as the one obtained with L1Md-A2 which is on average 95% homologous to the LINE-1 elements of the mouse. The endogenous LINE-1 sequences rescued by L1Rn-3 were 8-18% divergent from L1Rn-3 sequences, whereas those rescued by L1Md-A2 were 2-5% divergent from L1Md-A2 sequences. The gap which had been introduced into the exogenous LINE-1 sequences had been precisely repaired in 50% of the recombinants obtained with L1Md-A2. None of the L1Rn-3 recombinants showed precise gap repair.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of homology and pathway specificity for recombination between plasmids and bacteriophage lambda

Summary To determine the minimum amount of homology required for efficient recombination in Escherichia coli, we measured recombination frequencies between bacteriophage and pBR322 derivatives containing DNA fragments of various sizes by assaying for phages that could transduce the bla and ori genes of pBR322. Efficient recombination required about 40 bp of homology; increases in homology above 40 bp resulted in proportionate increases in recombination, while decreases below 40 bp resulted in precipitous decreases in recombination. The recA ⁺ gene stimulated recombination over the entire range of homologies tested. Restriction enzyme digests of several recombinant DNA molecules indicated that they contained the complete plasmid DNA inserted in the genome as expected for a reciprocal crossover. Analysis of recombination frequencies in different recombination-deficient mutant strains indicated that the formation of -plasmid cointegrates by homologous recombination proceeded predominantly by the RecBC pathway and very inefficiently, if at all, by the RecE and RecF pathways.     

Targeted modification of mammalian genomes

The stable and site-specific modification of mammalian genomes has a variety of applications in biomedicine and biotechnology. Here we outline two alternative approaches that can be employed to achieve this goal: homologous recombination (HR) or site-specific recombination. Homologous recombination relies on sequence similarity (or rather identity) of a piece of DNA that is introduced into a host cell and the host genome. In most cell types, the frequency of homologous recombination is markedly lower than the frequency of random integration. Especially in somatic cells, homologous recombination is an extremely rare event. However, recent strategies involving the introduction of DNA double-strand breaks, triplex forming oligonucleotides or adeno-associated virus can increase the frequency of homologous recombination.

Site-specific recombination makes use of enzymes (recombinases, transposases, integrases), which catalyse DNA strand exchange between DNA molecules that have only limited sequence homology. The recognition sites of site-specific recombinases (e.g. Cre, Flp or ΦC31 integrase) are usually 30–50 bp. In contrast, retroviral integrases only require a specific dinucleotide sequence to insert the viral cDNA into the host genome. Depending on the individual enzyme, there are either innumerable or very few potential target sites for a particular integrase/recombinase in a mammalian genome. A number of strategies have been utilised successfully to alter the site-specificity of recombinases. Therefore, site-specific recombinases provide an attractive tool for the targeted modification of mammalian genomes.