Efficient nonhomologous and homologous recombination in scid cells

The severe combined immunodeficiency (scid) mutation affects both coding joint formation during immunoglobulin and T-cell receptor V(D)J recombination and double-strand break repair. We analyzed scid cells for their ability to undergo other types of DNA end joining: non-homologous and homologous recombination. Using plasmid constructs carrying antibiotic resistance genes, we observed that the efficiency of nonhomologous integration in scid cells was equal to that in wildtype cell lines. In addition, there was no obvious difference in the fidelity of the integration and in the expression of the resistance genes. Moreover, scid cells were able to carry out homologous recombination of extrachromosomal substrates just as well as wildtype cells. These results suggest a mechanistic difference between nonhomologous integration and homologous recombination on the one hand and V(D)J recombination and double-strand break repair on the other.     

Biochemical studies of homologous and nonhomologous recombination in human cells.

Purified and partially purified protein fractions from human cells have been developed that promote homologous and nonhomologous recombination reactions in vitro. Homologous pairing of model DNA substrates is catalyzed by the homologous pairing protein HPP-1 in a magnesium-dependent, ATP-independent reaction that requires stoichiometric amounts of the protein. Addition of the human single-strand binding (SSB) holoprotein complex hRP-A reduces the requirement of HPP-1 in the reaction up to 20-fold. Although the combination of homologous pairing and SSB activities is similar to the bacterial strand-exchange process, the numbers, size, and requirements of the human reaction appear to preclude any detailed comparisons. We have used Z-DNA affinity chromatography as a major step in isolation of human recombination proteins and found that the activities appear to elute as a complex form in approximate multiples of 500 kDa. Associated with the homologous recombination complex is a potent blunt-end ligation activity that appears to mimic the nonhomologous joining functions that are frequently seen following transfection of DNA into mammalian cells. A simple scheme for the association of homologous and nonhomologous recombination functions in mammalian cells is discussed.     

Comparison of nonhomologous end joining and homologous recombination in human cells

The two major pathways for repair of DNA double-strand breaks (DSBs) are homologous recombination (HR) and nonhomologous end joining (NHEJ). HR leads to accurate repair, while NHEJ is intrinsically mutagenic. To understand human somatic mutation it is essential to know the relationship between these pathways in human cells. Here we provide a comparison of the kinetics and relative contributions of HR and NHEJ in normal human cells. We used chromosomally integrated fluorescent reporter substrates for real-time in vivo monitoring of the NHEJ and HR. By examining multiple integrated clones we show that the efficiency of NHEJ and HR is strongly influenced by chromosomal location. Furthermore, we show that NHEJ of compatible ends (NHEJ-C) and NHEJ of incompatible ends (NHEJ-I) are fast processes, which can be completed in approximately 30 min, while HR is much slower and takes 7h or longer to complete. In actively cycling cells NHEJ-C is twice as efficient as NHEJ-I, and NHEJ-I is three times more efficient than HR. Our results suggest that NHEJ is a faster and more efficient DSB repair pathway than HR.     

Alternative homologous and nonhomologous products arising from intramolecular recombination.

RmI, a chimeric DNA molecule containing polyomavirus (Py) and mouse sequences, generates unit-length Py DNA via intramolecular recombination between two directly repeated viral sequences of 182 base pairs (S repeats). To analyze the contribution of the S repeats in this process, we produced mutants of RmI carrying deletions in either one or both S repeats and tested them for their ability to recombine in mouse 3T6 cells. Mutant DNAs were found to yield unit-length Py DNA as long as they carried a minimal internal homology of 40 to 50 base pairs. Unlike RmI itself, however, the mutants also gave rise to nonhomologous recombination products. These results suggest that when the generation of homologous products is hampered by a limiting homology, nonhomologous products may arise instead of homologous ones. Therefore, the initial step(s) in the mechanisms yielding the two kinds of products could be identical.     

Insertion of nonhomologous DNA into the yeast genome mediated by homologous recombination with a cotransforming plasmid

Bacterial plasmids containing no detectable homology with yeast DNA sequences were inserted into the yeast genome by cotransforming with a plasmid containing a yeast gene. Analysis of the yeast transformants confirmed that recombination events occurred between the prokaryotic sequences shared by the two plasmids and between the yeast sequences common to the cotransforming plasmid and to the genome. Multiple copies of the two plasmids, in both tandem and interspersed arrays, are inserted by this method. Populations of cells grown from individual transformants are heterogenous for the number of integrated sequences. The number of integrated bacterial sequences is greatly reduced after 100 generations of growth in the populations that initially contained large numbers of sequences, while it is stable in those populations that initially contained either a single or a small number of copies.     

Transient stability of DNA ends allows nonhomologous end joining to precede homologous recombination

The stability of DNA ends generated by the HO endonuclease in yeast is surprisingly high with a half-life of more than an hour. This transient stability is unaffected by mutations that abolish nonhomologous end joining (NHEJ). The unprocessed ends interact with Yku70p and Yku80p, two proteins required for NHEJ, but not significantly with Rad52p, a protein involved in homologous recombination (HR). Repair of a double-strand break by NHEJ is unaffected by the possibility of HR, although the use of HR is increased in NHEJ-defective cells. Partial in vitro 5' strand processing suppresses NHEJ but not HR. These results show that NHEJ precedes HR temporally, and that the availability of substrate dictates the particular pathway used. We propose that transient stability of DNA ends is a foundation for the permanent stability of telomeres.     

Comparison of homologous recombination frequencies in somatic cells of petunia and tobacco suggest two distinct recombination pathways

Homologous recombination in plants was studied using an extrachromosomal recombination assay in which intermolecular homologous recombination between two complementary plasmids restored a selectable marker gene. Several vectors containing an insertion into or deletions within the coding region of the neomycin phosphotransferase (NPT-II) gene were designed. Plasmids were introduced, in pairwise combinations, into protoplasts and homologous recombination events were measured by counting the number of NPT-ll-resistant colonies. A 10-fold increase in recombination frequency was observed in Petunia hybrids RL01 compared to Nicotiana tabacum SRI. This difference occurred when one or both of the co-transferred recombination plasmids was offered in a circular form. Apart from such specific differences between two cultivars from different species, a two-to fivefold increase in recombination frequencies was observed when the genomic TBS (transformation booster sequence) fragment from P. hybrids was added onto one of the transferred plasmids. TBS-specific stimulation of recombination was observed in Petunia RL01. These data suggest that two different recombination pathways may be present in plants.     

Gene targeting in Aspergillus fumigatus by homologous recombination is facilitated in a nonhomologous end- joining-deficient genetic background

The akuA gene encoding the Ku70 component of the nonhomologous end-joining machinery was deleted in the opportunistic pathogen Aspergillus fumigatus. No obvious phenotype could be assessed for the corresponding mutant strain but relative frequencies of homologous recombination were increased as deduced from targeting the laccase-encoding abr2 gene.     

Different roles for nonhomologous end joining and homologous recombination following replication arrest in mammalian cells

Homologous recombination (HR) and nonhomologous end joining (NHEJ) play overlapping roles in repair of DNA double-strand breaks (DSBs) generated during the S phase of the cell cycle. Here, we characterized the involvement of HR and NHEJ in the rescue of DNA replication forks arrested or slowed by treatment of hamster cells with hydroxyurea or thymidine. We show that the arrest of replication with hydroxyurea generates DNA fragmentation as a consequence of the formation of DSBs at newly replicated DNA. Both HR and NHEJ protected cells from the lethal effects of hydroxyurea, and this agent also increased the frequency of recombination mediated by both homologous and nonhomologous exchanges. Thymidine induced a less stringent arrest of replication and did not generate detectable DSBs. HR alone rescued cells from the lethal effects of thymidine. Furthermore, thymidine increased the frequency of DNA exchange mediated solely by HR in the absence of detectable DSBs. Our data suggest that both NHEJ and HR are involved in repair of arrested replication forks that include a DSB, while HR alone is required for the repair of slowed replication forks in the absence of detectable DSBs.     

Unequal genetic exchange in Escherichia coli tandem duplications may represent a special pathway of homologous recombination

Heterozygous tandem duplications that appear in Escherichia coli conjugation matings segregate different types of haploid and diploid recombinants because of unequal crossing over between sister chromosomes. As shown previously, the frequency of segregants in the extended duplication D104 (approximately 150 kb or more than 3 min of the genetic map) heterozygous for E. coli deo-operon genes (deoA deoB::Tn5/deoC deoD) is not decreased in strains with defective RecBCD and RecF recombination pathways. Analysis of a shorter duplication of this type (approximately 46 kb) showed that the frequency of segregants in the strain recBC sbcBC recF was similar to that in a strain with undamaged system of recombination. Thus, genetic exchange between direct DNA repeats in tandem duplications may follow a special pathway of homologous recombination, which is independent of the recBC and recF genes.     

Genistein-induced DNA damage is repaired by nonhomologous end joining and homologous recombination in TK6 cells

Genistein (GES), a phytoestrogen, has potential chemopreventive and chemotherapeutic effects on cancer. The anticancer mechanism of GES may be related with topoisomerase II associated DNA double-strand breaks (DSBs). However, the precise molecular mechanism remains elusive. Here, we performed genetic analyses using human lymphoblastoid TK6 cell lines to investigate whether non-homologous DNA end joining (NHEJ) and homologous recombination (HR), the two major repair pathways of DSBs, were involved in repairing GES-induced DNA damage. Our results showed that GES induced DSBs in TK6 cells. Cells lacking Ligase4, an NHEJ enzyme, are hypersensitive to GES. Furthermore, the sensitivity of Ligase4^−/− cells was associated with enhanced DNA damage when comparing the accumulation of γ-H2AX foci and number of chromosomal aberrations (CAs) with WT cells. In addition, cells lacking Rad54, a HR enzyme, also presented hypersensitivity and increased DNA damages in response to GES. Meanwhile, Treatment of GES-lacking enhanced the accumulation of Rad51, an HR factor, in TK6 cells, especially in Ligase4^−/−. These results provided direct evidence that GES induced DSBs in TK6 cells and clarified that both NHEJ and HR were involved in the repair of GES-induced DNA damage, suggesting that GES in combination with inhibition of NHEJ or HR would provide a potential anticancer strategy.     

Role of Dnl4-Lif1 in nonhomologous end-joining repair complex assembly and suppression of homologous recombination

Nonhomologous end joining (NHEJ) eliminates DNA double-strand breaks (DSBs) in bacteria and eukaryotes. In Saccharomyces cerevisiae, there are pairwise physical interactions among the core complexes of the NHEJ pathway, namely Yku70-Yku80 (Ku), Dnl4-Lif1 and Mre11-Rad50-Xrs2 (MRX). However, MRX also has a key role in the repair of DSBs by homologous recombination (HR). Here we have examined the assembly of NHEJ complexes at DSBs biochemically and by chromatin immunoprecipitation. Ku first binds to the DNA end and then recruits Dnl4-Lif1. Notably, Dnl4-Lif1 stabilizes the binding of Ku to in vivo DSBs. Ku and Dnl4-Lif1 not only initiate formation of the nucleoprotein NHEJ complex but also attenuate HR by inhibiting DNA end resection. Therefore, Dnl4-Lif1 plays an important part in determining repair pathway choice by participating at an early stage of DSB engagement in addition to providing the DNA ligase activity that completes NHEJ.     

Quantitative multiplex real-time PCR for detection of PLP1 gene duplications in Pelizaeus-Merzbacher patients

Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive disorder of central nervous system (CNS) myelination typically affecting males. A genomic duplication of variable size at Xq22.2, containing the entire proteolipid protein 1 gene (PLP1), is responsible for approximately 60-70% of PMD cases. The aim of this study was to develop a rapid and robust method for determination of PLP1 gene dosage. We optimized two multiplex real-time quantitative PCR (Q-PCR) assays targeting exons 3 and 6 of the PLP1 gene, and then validated these assays by retrospective analysis of a set of genomic DNAs from 67 previously tested patients and 43 normal controls. Samples were analyzed in multiplex PCR reactions using TaqMan chemistry and the ABI Prism 7000 Sequence Detection System. PLP1 dosage was determined by the relative quantitative comparative threshold cycle method (DeltaDeltaCt) using the human serum albumin gene as the endogenous reference gene. Three clearly non-overlapping ranges of results, corresponding to the presence of one, two, or three PLP1 copies, were detected in both assays. The results were completely concordant with gender and previous PLP1 gene dosage testing based on quantitative fluorescent multiplex PCR and analysis of a dinucleotide polymorphism in the first intron of the PLP1 gene. We conclude that multiplex real-time Q-PCR represents a fast and reliable tool for PLP1 duplication testing in PMD families.     

Analysis of repair mechanism choice during homologous recombination

Double-strand breaks (DSBs) occur frequently during cell growth. Due to the presence of repeated sequences in the genome, repair of a single DSB can result in gene conversion, translocation, deletion or tandem duplication depending on the mechanism and the sequence chosen as partner for the recombinational repair. Here, we study how yeast cells repair a single, inducible DSB when there are several potential donors to choose from, in the same chromosome and elsewhere in the genome. We systematically investigate the parameters that affect the choice of mechanism, as well as its genetic regulation. Our results indicate that intrachromosomal homologous sequences are always preferred as donors for repair. We demonstrate the occurrence of a novel tri-partite repair product that combines ectopic gene conversion and deletion. In addition, we show that increasing the distance between two repeated sequences enhances the dependence on Rad51 for colony formation after DSB repair. This is due to a role of Rad51 in the recovery from the checkpoint signal induced by the DSB. We suggest a model for the competition between the different homologous recombination pathways. Our model explains how different repair mechanisms are able to compensate for each other during DSB repair.     

The high variability of subtelomeric heterochromatin and connections between nonhomologous chromosomes, suggest frequent ectopic recombination in rye meiocytes

The position of telomeres, centromeres and subtelomeric heterochromatin (SH) has been studied by FISH in rye meiocytes. We compare the morphology of the signals from zygotene to telophase II mainly to determine differences in SH and telomere positions between plants with and without neocentromeres. Plants from two varieties were used: Paldang showing neocentromeres, and Puyo without neocentromeres but with two B chromosomes. In both varieties, at zygotene and pachytene the SH is observed forming clumps often including two or more bivalent ends. At diplotene the SH is stretched suggesting that it is close to the nuclear envelope. In these cases, the telomere signals are not stretched and lay behind the SH. Frequently, two or more bivalents are joined by conspicuous SH connections at diplotene strongly suggesting ectopic recombination. Probably as a result, differential distribution of the SH between recombinant homologues or the whole meiotic products is observed. From diplotene onwards, the large heterochromatic blocks cover the telomeres, the SH being the morphological end of the bivalents, both in plants with or without neocentromeres. The Bs are tightly associated only at the telomeric end of the long arm from diplotene to metaphase I. The high variability between homologous chromosomes and the frequent nonhomologous bindings of SH, strongly suggest that rye SH is in dynamic state and frequently changes in chromosome position during meiosis.     

Intermolecular recombination assay for mammalian cells that produces recombinants carrying both homologous and nonhomologous junctions.

We present an intermolecular recombination assay for mammalian cells that does not involve the reconstitution of a selectable marker. It is based on the generation of a shuttle vector by recombination between a bacterial and a mammalian vector. The recombinants can thus be amplified in mammalian cells, isolated by plasmid rescue in an Escherichia coli RecA- host, and identified by in situ hybridization, by using mammalian vector sequences as probes. Since both parental molecules can share defined lengths of homology, this assay permits a direct comparison between homologous and nonhomologous intermolecular recombination. Our results indicate that the dominant intermolecular recombination mechanism is a nonhomologous one. The relative frequency of homologous to nonhomologous recombination was influenced by the length of shared homology between parental molecules and the replicative state of the parental molecules, but not by the introduction of double-strand breaks per se. Finally, almost all of the recombinants with a homologous junction did not have the reciprocal homologous junction but instead had a nonhomologous one. We propose a model to account for the generation of these recombinants.     

High-frequency homologous plasmid-plasmid recombination coupled with conjugation of plasmid SCP2* in Streptomyces

Non-transmissible derivatives of the Streptomyces multi-copy plasmid pIJ101 were mobilized, by co-integrate formation, at frequencies approaching 100% (measured per recipient) by derivatives of the conjugative, low-copy-number Streptomyces coeli-color A3(2) plasmid SCP2*. Efficient co-integrate formation required that the plasmids shared at least 112 bp sequence identity, and it occurred only during conjugation. An SCP2* plasmid gene is involved in the process. Co-integrates were presumably formed in the donor cells and transported to the recipient cells. This is a new phenomenon, not known in other bacteria.     

Mechanisms of tandem duplication in the Duchenne muscular dystrophy gene include both homologous and nonhomologous intrachromosomal recombination.

Three tandem duplications were previously identified in patients with Duchenne muscular dystrophy and were shown in each case to have a subset of dystrophin gene exons duplicated. The origin of these duplications was traced to the single X chromosome of the maternal grandfathers, suggesting that an intrachromosomal event (unequal sister chromatid exchange) was involved in the formation of these duplications. In the present study, a DNA segment containing the duplication junction and the normal DNA that corresponds to both ends of the duplicated region have been cloned. Subsequent mapping studies confirmed the tandem arrangement (head to tail) of these duplications and revealed their sizes to be 130 kb, approximately 300 kb, and 35-80 kb, respectively. Sequence analysis of the duplication junctions showed that one duplication was due to homologous recombination between two repetitive elements (Alu sequences) and the other two were due to recombination between unrelated nonhomologous sequences. In the latter cases, the preferred cleavage sites of the eukaryotic type I and II DNA topoisomerases were found at the junctions of these duplications, suggesting a possible role of these enzymes in the chromatid exchange events. This study provides the first insight into the molecular basis of gene duplications formed through unequal sister chromatid exchange in humans.     

Different mechanisms of homologous and nonhomologous recombination in brome mosaic virus: role of RNA sequences and replicase proteins

Brome mosaic bromovirus, a tripartite, positive-stranded RNA virus of plants, can generate both homologous and nonhomologous recombinantsin vivo. Recombination signals in the RNAs were different for these two recombination types. Nonhomologous recombination requires the formation of local double-stranded regions between the RNA templates. In contrast, homologous recombination is facilitated by AU-rich sequences and upstream GC-rich regions common in the recombining RNAs. Mutations within the replicase proteins affect homologous and nonhomologous recombination in different ways, confirming the involvement of BMV replicase proteins in both types of events as well as the differences in their pathways. Replicase-driven template-switching models are discussed in relation to supporting evidences.     

Unequal crossing over is the principal pathway of homologous recombination in tandem duplications of Escherichia coli

Homologous recombination between direct DNA repeats in tandem duplications usually leads to their dissociation. An even number of crossovers between two copies of a duplication should lead to the formation of diploid segregants, i.e., to the preservation of the duplication. However, in studies of the genotype of diploid segregants in heterozygous tandem duplications of Escherichia coli, it was shown that they arise by unequal exchanges between sister chromosomes rather than by intrachromosomal exchanges. Generally, these exchanges lead to the establishment of the homozygous state of (heterozygous) duplications. Since the available data suggest that the exchange between sister chromosomes may be coupled with DNA replication, it is supposed that unequal exchanges between direct DNA repeats occur in the process of DNA replication.