Effect of DNA sequence divergence on homologous recombination as analyzed by a random-walk model

A point connecting a pair of homologous regions of DNA duplexes moves along the homology in a reaction intermediate of the homologous recombination. Formulating this movement as a random walk, we were previously successful at explaining the dependence of the recombination frequency on the homology length. Recently, the dependence of the recombination frequency on the DNA sequence divergence in the homologous region was investigated experimentally; if the methyl-directed mismatch repair (MMR) system is active, the logarithm of the recombination frequency decreases very rapidly with an increase of the divergence in a low-divergence regime. Beyond this regime, the logarithm decreases slowly and linearly with the divergence. This "very rapid drop-off" is not observed when the MMR system is defective. In this article, we show that our random-walk model can explain these data in a straightforward way. When a connecting point encounters a diverged base pair, it is assumed to be destroyed with a probability that depends on the level of MMR activity.     

A conditional mouse model for measuring the frequency of homologous recombination events in vivo in the absence of essential genes

The ability to detect and repair DNA damage is crucial to the prevention of various diseases. Loss of function of genes involved in these processes is known to result in significant developmental defects and/or predisposition to cancer. One such DNA repair mechanism, homologous recombination, has the capacity to repair a wide variety of lesions. Knockout mouse models of genes thought to be involved in DNA repair processes are frequently lethal, making in vivo studies very difficult, if not impossible. Therefore, we set out to develop an in vivo conditional mouse model system to facilitate investigations into the involvement of essential genes in homologous recombination. To test our model, we measured the frequency of spontaneous homologous recombination using the pink-eyed unstable mouse model, in which we conditionally excised either Blm or full-length Brca1 (breast cancer 1, early onset). These two genes are hypothesized to have opposing roles in homologous recombination. In summary, our in vivo data supports in vitro studies suggesting that BLM suppresses homologous recombination, while full-length BRCA1 promotes this process.     

Abiotic stress leads to somatic and heritable changes in homologous recombination frequency, point mutation frequency and microsatellite stability in Arabidopsis plants

In earlier studies, we showed that abiotic stresses, such as ionizing radiation, heavy metals, temperature and water, trigger an increase in homologous recombination frequency (HRF). We also demonstrated that many of these stresses led to inheritance of high-frequency homologous recombination, HRF. Although an increase in recombination frequency is an important indicator of genome rearrangements, it only represents a minor portion of possible stress-induced mutations. Here, we analyzed the influence of heat, cold, drought, flood and UVC abiotic stresses on two major types of mutations in the genome, point mutations and small deletions/insertions. We used two transgenic lines of Arabidopsis thaliana, one allowing an analysis of reversions in a stop codon-containing inactivated β-glucuronidase transgene and another one allowing an analysis of repeat stability in a microsatellite-interrupted β-glucuronidase transgene. The transgenic Arabidopsis line carrying the β-glucuronidase-based homologous recombination substrate was used as a positive control. We showed that the majority of stresses increased the frequency of point mutations, homologous recombination and microsatellite instability in somatic cells, with the frequency of homologous recombination being affected the most. The analysis of transgenerational changes showed an increase in HRF to be the most prominent effect observed in progeny. Significant changes in recombination frequency were observed upon exposure to all types of stress except drought, whereas changes in microsatellite instability were observed upon exposure to UVC, heat and cold. The frequency of point mutations in the progeny of stress-exposed plants was the least affected; an increase in mutation frequency was observed only in the progeny of plants exposed to UVC. We thus conclude that transgenerational changes in genome stability in response to stress primarily involve an increase in recombination frequency.     

Duration time of a one-dimensional random walk as a function of the energies of the intermediate states: application for dissociation and relaxation processes in DNA hybrids.

Kinetic parameters of macromolecular systems are important for their function in vitro and in vivo. These parameters describe how fast the system dissociates (the characteristic dissociation time), and how fast the system reaches equilibrium (characteristic relaxation time). For many macromolecular systems, the transitions within the systems are described as a random walk through a number of states with various free energies. The rate of transition between two given states within the system is characterized by the average time which passes between starting the movement from one state, and reaching the other state. This time is referred to as the mean first-passage time between two given states. The characteristic dissociation and relaxation times of the system depend on the first-passages times between the states within the system. Here, for a one-dimensional random walk we derived an equation, which connects the mean first-passage time between two states with the free energies of the states within the system. We also derived the general equation, which is not restricted to one-dimensional systems, connecting the relaxation time of the system with the first-passage times between states. The application of these equations to DNA branch migration, DNA structural transitions and other processes is discussed.     

In vivo recombination after chronic damage exposure falls to below spontaneous levels in "recombomice"

All forms of cancer are initiated by heritable changes in gene expression. Although point mutations have been studied extensively, much less is known about homologous recombination events, despite its role in causing sequence rearrangements that contribute to tumorigenesis. Although transgenic mice that permit detection of point mutations have provided a fundamental tool for studying point mutations in vivo, until recently, transgenic mice designed specifically to detect homologous recombination events in somatic tissues in vivo did not exist. We therefore created fluorescent yellow direct repeat mice, enabling automated detection of recombinant cells in vivo for the first time. Here, we show that an acute dose of ionizing radiation induces recombination in fluorescent yellow direct repeat mice, providing some of the first direct evidence that ionizing radiation induces homologous recombination in cutaneous tissues in vivo. In contrast, the same total dose of radiation given under chronic exposure conditions suppresses recombination to levels that are significantly below those of unexposed animals. In addition, global methylation is suppressed and key DNA repair proteins are induced in tissues from chronically irradiated animals (specifically AP endonuclease, polymerase beta, and Ku70). Thus, increased clearance of recombinogenic lesions may contribute to suppression of homologous recombination. Taken together, these studies show that fluorescent yellow direct repeat mice provide a rapid and powerful assay for studying the recombinogenic effects of both short-term and long-term exposure to DNA damage in vivo and reveal for the first time that exposure to ionizing radiation can have opposite effects on genomic stability depending on the duration of exposure.     

Visualizing the Behavior of Human Rad51 at the Single-Molecule Level

The repair of double-stranded DNA breaks by homologous recombination is essential for maintaining genome integrity. Much of what we know about this DNA repair pathway in eukaryotes has been gleaned from genetics, in vivo experiments with GFP-tagged proteins and traditional biochemical experiments with purified proteins. However, many questions have remained inaccessible to these experimental approaches. Recent technological advances have made it possible to directly visualize the behaviors of individual DNA and protein molecules in vitro, and it is now becoming feasible to apply these technology-driven approaches to complex biochemical systems, such as those involved in the repair of damaged DNA. This report summarizes the use of total internal reflection fluorescence microscopy to probe fundamental aspects of protein-DNA interactions at the single-molecule level, and specific emphasis is placed on our efforts to develop new methods and techniques for studying DNA repair. Using these new approaches we are investigating the DNA-binding behavior of human Rad51 and we have recently demonstrated that this protein can slide on dsDNA via a one-dimensional random walk mechanism driven solely by thermal fluctuations of the surrounding solvent. Here, we highlight some possible implications of this recent finding, and we also briefly discuss the potential benefits of future single-molecule studies in the study of protein-DNA interactions and DNA repair.     

Efficient in vitro system of homologous recombination in brome mosaic bromovirus

Recent in vivo studies have revealed that the subgenomic promoter (sgp) in brome mosaic bromovirus (BMV) RNA3 supports frequent homologous recombination events (R. Wierzchoslawski, A. Dzianott, and J. Bujarski, J. Virol. 78:8552-8564, 2004). In this paper, we describe an sgp-driven in vitro system that supports efficient RNA3 crossovers. A 1:1 mixture of two (-)-sense RNA3 templates was copied with either a BMV replicase (RdRp) preparation or recombinant BMV protein 2a. The BMV replicase enzyme supported a lower recombination frequency than 2a, demonstrating a role of other viral and/or host factors. The described in vitro system will allow us to study the mechanism of homologous RNA 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.     

Protein-primed DNA replication: role of inverted terminal repeats in the Escherichia coli bacteriophage PRD1 life cycle.

Escherichia coli bacteriophage PRD1 and its relatives contain linear double-stranded DNA genomes, the replication of which proceeds via a protein-primed mechanism. Characteristically, these molecules contain 5'-covalently bound terminal proteins and inverted terminal nucleotide sequences (inverted terminal repeats [ITRs]). The ITRs of each PRD1 phage species have evolved in parallel, suggesting communication between the molecule ends during the life cycle of these viruses. This process was studied by constructing chimeric PRD1 phage DNA molecules with dissimilar end sequences. These molecules were created by combining two closely related phage genomes (i) in vivo by homologous recombination and (ii) in vitro by ligation of appropriate DNA restriction fragments. The fate of the ITRs after propagation of single genomes was monitored by DNA sequence analysis. Recombinants created in vivo showed that phages with nonidentical genome termini are viable and relatively stable, and hybrid phages made in vitro verified this observation. However, genomes in which the dissimilar DNA termini had regained identical sequences were also detected. These observations are explained by a DNA replication model involving two not mutually exclusive pathways. The generality of this model in protein-primed DNA replication is discussed.     

Purification of the human SMN-GEMIN2 complex and assessment of its stimulation of RAD51-mediated DNA recombination reactions

A deficiency in the SMN gene product causes the motor neuron degenerative disease spinal muscular atrophy. GEMIN2 was identified as an SMN-interacting protein, and the SMN-GEMIN2 complex constitutes part of the large SMN complex, which promotes the assembly of the spliceosomal small nuclear ribonucleoprotein (snRNP). In addition to its splicing function, we previously found that GEMIN2 alone stimulates RAD51-mediated recombination in vitro, and functions in DNA double-strand-break (DSB) repair through homologous recombination in vivo. However, the function of SMN in homologous recombination has not been reported. In the present study, we successfully purified the SMN-GEMIN2 complex as a fusion protein. The SMN-GEMIN2 fusion protein complemented the growth-defective phenotype of GEMIN2-knockout cells. The purified SMN-GEMIN2 fusion protein enhanced the RAD51-mediated homologous pairing much more efficiently than GEMIN2 alone. SMN-GEMIN2 possessed DNA-binding activity, which was not observed with the GEMIN2 protein, and significantly stimulated the secondary duplex DNA capture by the RAD51-single-stranded DNA complex during homologous pairing. These results provide the first evidence that the SMN-GEMIN2 complex plays a role in homologous recombination, in addition to spliceosomal snRNP assembly.     

Homologous recombination in a Chinese hamster X-ray-sensitive mutant

We have tested the mutant Chinese hamster cell line xrs-5, which is sensitive to ionizing radiation, for the ability to carry out homologous recombination. In an in vivo assay to detect recombination between two transfected plasmids carrying non-complementing mutants in the neomycin resistance gene, xrs-5 showed a 6-fold reduction in recombination frequency when compared to the parental cell line K1. Extracts prepared from nuclei of the mutant were also tested for their ability to catalyze homologous recombination between the same two plasmids in vitro. Extracts from xrs-5 were found to mediate recombination in this assay at frequencies not significantly different from those obtained with extracts from the parental cell line.     

Single Holliday junctions are intermediates of meiotic recombination

Crossing-over between homologous chromosomes facilitates their accurate segregation at the first division of meiosis. Current models for crossing-over invoke an intermediate in which homologs are connected by two crossed-strand structures called Holliday junctions. Such double Holliday junctions are a prominent intermediate in Saccharomyces cerevisiae meiosis, where they form preferentially between homologs rather than between sister chromatids. In sharp contrast, we find that single Holliday junctions are the predominant intermediate in Schizosaccharomyces pombe meiosis. Furthermore, these single Holliday junctions arise preferentially between sister chromatids rather than between homologs. We show that Mus81 is required for Holliday junction resolution, providing further in vivo evidence that the structure-specific endonuclease Mus81-Eme1 is a Holliday junction resolvase. To reconcile these observations, we present a unifying recombination model applicable for both meiosis and mitosis in which single Holliday junctions arise from single- or double-strand breaks, lesions postulated by previous models to initiate recombination.     

A Sister-Strand Exchange Mechanism for Reca-Independent Deletion of Repeated DNA Sequences in Escherichia Coli

In the genomes of many organisms, deletions arise between tandemly repeated DNA sequences of lengths ranging from several kilobases to only a few nucleotides. Using a plasmid-based assay for deletion of a 787-bp tandem repeat, we have found that a recA-independent mechanism contributes substantially to the deletion process of even this large region of homology. No Escherichia coli recombination gene tested, including recA, had greater than a fivefold effect on deletion rates. The recA-independence of deletion formation is also observed with constructions present on the chromosome. RecA promotes synapsis and transfer of homologous DNA strands in vitro and is indispensable for intermolecular recombination events in vivo measured after conjugation. Because deletion formation in E. coli shows little or no dependence on recA, it has been assumed that homologous recombination contributes little to the deletion process. However, we have found recA-independent deletion products suggestive of reciprocal crossovers when branch migration in the cell is inhibited by a ruvA mutation. We propose a model for recA-independent crossovers between replicating sister strands, which can also explain deletion or amplification of repeated sequences. We suggest that this process may be initiated as post-replicational DNA repair; subsequent strand misalignment at repeated sequences leads to genetic rearrangements.     

Antipairing and strand transferase activities of E. coli helicase II (UvrD).

The product of the uvrD gene of Escherichia coli, UvrD (helicase II), is known to be involved in methyl-directed mismatch repair, transposon excision and uvrABC excision repair. In conjugational crosses, various uvrD mutants have been reported to result in higher, lower or unaffected recombination frequencies. In an attempt to clarify the role of UvrD in recombination, we have studied in vitro its effects on two key reactions driven by RecA, homologous pairing and strand exchange. We show here that UvrD efficiently prevents or reverses RecA-mediated homologous pairing. Unexpectedly, we also found that it can stimulate RecA-driven branch migration and even catalyze strand exchange in the absence of RecA. A possible in vivo role for these antagonistic activities is discussed.     

p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction.

The tumour suppressor p53 prevents tumour formation after DNA damage by halting cell cycle progression to allow DNA repair or by inducing apoptotic cell death. Loss of wild-type p53 function renders cells resistant to DNA damage-induced cell cycle arrest and ultimately leads to genomic instabilities including gene amplifications, translocations and aneuploidy. Some of these chromosomal lesions are based on mechanisms that involve recombinatorial events. Here we report that p53 physically interacts with key factors of homologous recombination: the human RAD51 protein and its prokaryotic homologue RecA. In vitro, wild-type p53 inhibits defined biochemical activities of RecA protein, such as three-way DNA strand exchange and single strand DNA-dependent ATPase activity. In vivo, temperature-sensitive p53 forms complexes with RAD51 only in wild-type but not in mutant conformation. These observations suggest that functional wild-type p53 may select directly the appropriate pathway for DNA repair and control the extent and timing of the production of genetic variation via homologous recombination. Gene amplification an other types of chromosome rearrangements involved in tumour progression might occur not only as result of inappropriate cell proliferation but as a direct consequence of a defect in p53-mediated control of homologous recombination processes due to mutations in the p53 gene.     

Random Field Model Reveals Structure of the Protein Recombinational Landscape

We are interested in how intragenic recombination contributes to the evolution of proteins and how this mechanism complements and enhances the diversity generated by random mutation. Experiments have revealed that proteins are highly tolerant to recombination with homologous sequences (mutation by recombination is conservative); more surprisingly, they have also shown that homologous sequence fragments make largely additive contributions to biophysical properties such as stability. Here, we develop a random field model to describe the statistical features of the subset of protein space accessible by recombination, which we refer to as the recombinational landscape. This model shows quantitative agreement with experimental results compiled from eight libraries of proteins that were generated by recombining gene fragments from homologous proteins. The model reveals a recombinational landscape that is highly enriched in functional sequences, with properties dominated by a large-scale additive structure. It also quantifies the relative contributions of parent sequence identity, crossover locations, and protein fold to the tolerance of proteins to recombination. Intragenic recombination explores a unique subset of sequence space that promotes rapid molecular diversification and functional adaptation.     

Evidence for the murine IgH mu locus acting as a hot spot for intrachromosomal homologous recombination

Homologous recombination accomplishes the exchange of genetic information between two similar or identical DNA duplexes. It can occur either by gene conversion, a process of unidirectional genetic exchange, or by reciprocal crossing over. Homologous recombination is well known for its role in generating genetic diversity in meiosis and, in mitosis, as a DNA repair mechanism. In the immune system, the evidence suggests a role for homologous recombination in Ig gene evolution and in the diversification of Ab function. Previously, we reported the occurrence of homologous recombination between repeated, donor and recipient alleles of the Ig H chain mu gene C (Cmu) region residing at the Ig mu locus in mouse hybridoma cells. In this study, we constructed mouse hybridoma cell lines bearing Cmu region heteroalleles to learn more about the intrachromosomal homologous recombination process. A high frequency of homologous recombination (gene conversion) was observed for markers spanning the entire recipient Cmu region, suggesting that recombination might initiate at random sites within the Cmu region. The Cmu region heteroalleles were equally proficient as either conversion donors or recipients. Remarkably, when the same Cmu heteroalleles were tested for recombination in ectopic genomic positions, the mean frequency of gene conversion was reduced by at least 65-fold. These results are consistent with the murine IgH mu locus behaving as a hot spot for intrachromosomal homologous recombination.     

A new type of illegitimate recombination is dependent on restriction and homologous interaction.

Illegitimate (nonhomologous) 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. Under special conditions in Escherichia coli, we have found a new type of illegitimate recombination that requires an interaction between homologous DNA sequences. It was detected when a plasmid that carried 2-kb-long inverted repeats was subjected to type II restriction in vitro and type I (EcoKI) restriction in vivo within a delta rac recBC recG ruvC strain. Removal of one of the repeats or its replacement with heterologous DNA resulted in a reduction in the level of recombination. The recombining sites themselves shared, at most, a few base pairs of homology. Many of the recombination events joined a site in one of the repeats with a site in another repeat. In two of the products, one of the recombining sites was at the end of one of the repeats. Removal of one of the EcoKI sites resulted in decreased recombination. We discuss the possibility that some structure made by homologous interaction between the long repeats is used by the EcoKI restriction enzyme to promote illegitimate recombination. The possible roles and consequences of this type of homologous interaction are discussed.     

Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells.

Cultured animal cells rearrange foreign DNA very efficiently by homologous recombination. The individual steps that constitute the mechanism(s) of homologous recombination in transfected DNA are as yet undefined. In this study, we examined the topological requirements by using the genome of simian virus 40 (SV40) as a probe. By assaying homologous recombination between defective SV40 genomes after transfection into CV1 monkey cells, we showed that linear molecules are preferred substrates for homologous exchanges, exchanges are distributed around the SV40 genome, and the frequency of exchange is not diminished significantly by the presence of short stretches of non-SV40 DNA at the ends. These observations are considered in relation to current models of homologous recombination in mammalian cells, and a new model is proposed. The function of somatic cell recombination is discussed.     

Increased chromosome mobility facilitates homology search during recombination

Homologous recombination, an essential process for preserving genomic integrity, uses intact homologous sequences to repair broken chromosomes. To explore the mechanism of homologous pairing in vivo, we tagged two homologous loci in diploid yeast Saccharomyces cerevisiae cells and investigated their dynamic organization in the absence and presence of DNA damage. When neither locus is damaged, homologous loci occupy largely separate regions, exploring only 2.7% of the nuclear volume. Following the induction of a double-strand break, homologous loci co-localize ten times more often. The mobility of the cut chromosome markedly increases, allowing it to explore a nuclear volume that is more than ten times larger. Interestingly, the mobility of uncut chromosomes also increases, allowing them to explore a four times larger volume. We propose a model for homology search in which increased chromosome mobility facilitates homologous pairing. Finally, we find that the increase in DNA dynamics is dependent on early steps of homologous recombination.