Xrcc2 is required for genetic stability, embryonic neurogenesis and viability in mice

Repair of DNA damage by homologous recombination has only recently been established as an important mechanism in maintaining genetic stability in mammalian cells. The recently cloned Xrcc2 gene is a member of the mammalian Rad51 gene family, thought to be central to homologous recombination repair. To understand its function in mammals, we have disrupted Xrcc2 in mice. No Xrcc2(-/-) animals were found alive, with embryonic lethality occurring from mid-gestation. Xrcc2(-/-) embryos surviving until later stages of embryogenesis commonly showed developmental abnormalities and died at birth. Neonatal lethality, apparently due to respiratory failure, was associated with a high frequency of apoptotic death of post- mitotic neurons in the developing brain, leading to abnormal cortical structure. Embryonic cells showed genetic instability, revealed by a high level of chromosomal aberrations, and were sensitive to gamma-rays. Our findings demonstrate that homologous recombination has an important role in endogenous damage repair in the developing embryo. Xrcc2 disruption identifies a range of defects that arise from malfunction of this repair pathway, and establishes a previously unidentified role for homologous recombination repair in correct neuronal development.     

Properties of natural double-strand-break sites at a recombination hotspot in Saccharomyces cerevisiae

This study addresses three questions about the properties of recombination hotspots in Saccharomyces cerevisiae: How much DNA is required for double-strand-break (DSB) site recognition? Do naturally occurring DSB sites compete with each other in meiotic recombination? What role does the sequence located at the sites of DSBs play? In S. cerevisiae, the HIS2 meiotic recombination hotspot displays a high level of gene conversion, a 3''-to-5'' conversion gradient, and two DSB sites located approximately 550 bp apart. Previous studies of hotspots, including HIS2, suggest that global chromosome structure plays a significant role in recombination activity, raising the question of how much DNA is sufficient for hotspot activity. We find that 11.5 kbp of the HIS2 region is sufficient to partially restore gene conversion and both DSBs when moved to another yeast chromosome. Using a variety of different constructs, studies of hotspots have indicated that DSB sites compete with one another for DSB formation. The two naturally occurring DSBs at HIS2 afforded us the opportunity to examine whether or not competition occurs between these native DSB sites. Small deletions of DNA at each DSB site affect only that site; analyses of these deletions show no competition occurring in cis or in trans, indicating that DSB formation at each site at HIS2 is independent. These small deletions significantly affect the frequency of DSB formation at the sites, indicating that the DNA sequence located at a DSB site can play an important role in recombination initiation.     

DNA-dependent protein kinase mediates V(D)J recombination via RAG2 phosphorylation

V(D)J recombination, a site-specific gene rearrangement process occurring during the lymphocyte development, begins with DNA double strand breaks by two recombination activating gene products (RAG1/2) and finishes with the repair process by several proteins including DNA-dependent protein kinase (DNA-PK). In this report, we found that RAG2 was specifically phosphorylated by DNA-PK at the 365(th) serine residue, and this phosphorylated RAG2 affected the V(D)J recombination activity in cells in the GFP expression-based assay. While the V(D)J recombination activity between wild-type RAG2 and mutant S365A RAG2 in the assay using a signal joint substrate was undistinguishable in DNA-PK deficient cells (M059J), the activity with wild-type RAG2 was largely increased in DNA-PK proficient cells (M059K) in comparison with mutant RAG2, suggesting that RAG2 phosphorylation by DNA-PK plays a crucial role in the signal joint formation during V(D)J recombination.     

Recombination occurs uniformly within the bronze gene, a meiotic recombination hotspot in the maize genome.

The bronze (bz) gene is a recombinational hotspot in the maize genome: its level of meiotic recombination per unit of physical length is > 100-fold higher than the genome's average and is the highest of any plant gene analyzed to date. Here, we examine whether recombination is also unevenly distributed within the bz gene. In yeast genes, recombination (conversion) is polarized, being higher at the end of the gene where recombination is presumably initiated. We have analyzed products of meiotic recombination between heteroallelic pairs of bz mutations in both the presence and absence of heterologies and have sequenced the recombination junction in 130 such Bz intragenic recombinants. We have found that in the absence of heterologies, recombination is proportional to physical distance across the bz gene. The simplest interpretation for this lack of polarity is that recombination is initiated randomly within the gene. Insertion mutations affect the frequency and distribution of intragenic recombination events at bz, creating hotspots and coldspots. Single base pair heterologies also affect recombination, with fewer recombination events than expected by chance occurring in regions of the bz gene with a high density of heterologies. We also provide evidence that meiotic recombination in maize is conservative, that is, it does not introduce changes, and that meiotic conversion tracts are continuous and similar in size to those in yeast.     

Comparative analysis of nuclear proteins of B cells in different developmental stages

The developmental stage-specific regulation of V(D)J recombination, a gene rearrangement process of antigen receptor gene segments, is tightly controlled in cells. Here we screened proteins uniquely or differentially expressed among three developmentally distinguishable B cells (pro-B, pre-B and mature B cells) using two-dimensional gel electrophoresis and mass spectrometry. Chromatin assembly factor 1 was uniquely expressed in pro-B cells. Purine nucleotide phosphorylase, LCK, E2A and many other unidentified proteins were dominantly present in the nucleus at the early stage of B cell development where the V(D)J recombination process occurs. Also, few proteins including guanidine nucleotide binding proteins were exclusively expressed in pre-B cell. Such findings can provide some information to help understand the developmental regulation of gene rearrangement occurring during B cell development.     

Allelic dimorphism-associated restriction of recombination in Plasmodium falciparum msp1

Allelic dimorphism is a characteristic feature of the Plasmodium falciparum msp1 gene encoding the merozoite surface protein 1, a strong malaria vaccine candidate. Meiotic recombination is a major mechanism for the generation of msp1 allelic diversity. Potential recombination sites have previously been mapped to specific regions within msp1 (a 5' 1-kb region and a 3' 0.4-kb region) with no evidence for recombination events in a central 3.5-kb region. However, evidence for the lack of recombination events is circumstantial and inconclusive because the number of msp1 sequences analysed is limited, and the frequency of recombination events has not been addressed previously in a high transmission area, where the frequency of meiotic recombination is expected to be high. In the present study, we have mapped potential allelic recombination sites in 34 full-length msp1 sequences, including 24 new sequences, from various geographic origins. We also investigated recombination events in blocks 6 to 16 by population genetic analysis of P. falciparum populations in Tanzania, where malaria transmission is intense. The results clearly provide no evidence of recombination events occurring between the two major msp1 allelic types, K1-type and Mad20-type, in the central region, but do show recombination events occurring throughout the entire gene within sequences of the Mad20-type. Thus, the present study indicates that allelic dimorphism of msp1 greatly affects inter-allelic recombination events, highlighting a unique feature of allelic diversity of P. falciparum msp1.     

甲型流行性感冒病毒自然温度敏感株基因损害定位

采用重组试验和聚丙烯酰胺凝胶电泳(PAGE)技术,把晚期甲3型流感病毒自然ts突变株齐防79-39的ts损害定位在膜蛋白(M)基因上。但互补试验表明,齐防79-39与M基因损害的WSN标准株ts51可以发生互补,这是基因内互补的一个证据。PAGE技术证实,新甲1型流感病毒自然ts株津防77-78的M基因上确有损害。互补试验证明齐防79-39属于一个互补组,而津防77-78与ts51同属于另一个互补组。 本文结果还表明,晚期甲3型齐防79-39的ts损害基因可能是由甲3型野毒株自发突变所产生,而并非通过在自然界与新甲1型重组而获得。     

Evidence that recombination between reiterated sequences in the Bacillus subtilis chromosome does not occur via unequal crossing over.

An experimental system was designed to permit the detection of recombination events occurring via unequal crossing over between sister bacterial chromosomes in Bacillus subtilis. It exploits the fact that during spore development, genetic and metabolic cooperation occurs between two different cell types, only one of which survives. During the early stages of sporulation, the two chromosomes of the developing sporangiole lie in the same cell and recombination between them is possible, in principle. Internal duplications flanking a selectable antibiotic-resistance gene have been introduced into the spoIIIC, spoIVA and spoVJ genes, whose correct expression in the mother cell (non-surviving compartment) is necessary for completion of spore development. After incubation in a sporulation-inducing medium in the absence of selective pressure, these strains sporulate at a low frequency and up to 30% of the progeny are Spo-. They result from mosaic sporangioles, in which only the chromosome segregated into the mother cell compartment of the developing sporangiole contains a reconstituted spo gene. In mosaic sporangioles generated by unequal crossing over between sister bacterial chromosomes, the insertionally inactivated spo gene, segregated into the pre-spore compartment, would carry an extra copy of the duplication initially present. Analysis of the products of 124 independent recombination events giving rise to mosaic sporangioles provided no evidence for the occurrence of unequal crossing over.     

Replication fork blockage by RTS1 at an ectopic site promotes recombination in fission yeast

Homologous recombination is believed to play important roles in processing stalled/blocked replication forks in eukaryotes. In accordance with this, recombination is induced by replication fork barriers (RFBs) within the rDNA locus. However, the rDNA locus is a specialised region of the genome, and therefore the action of recombinases at its RFBs may be atypical. We show here for the first time that direct repeat recombination, dependent on Rad22 and Rhp51, is induced by replication fork blockage at a site-specific RFB (RTS1) within a 'typical' genomic locus in fission yeast. Importantly, when the RFB is positioned between the direct repeat, conservative gene conversion events predominate over deletion events. This is consistent with recombination occurring without breakage of the blocked fork. In the absence of the RecQ family DNA helicase Rqh1, deletion events increase dramatically, which correlates with the detection of one-sided DNA double-strand breaks at or near RTS1. These data indicate that Rqh1 acts to prevent blocked replication forks from collapsing and thereby inducing deletion events.     

Accurately Measuring Recombination between Closely Related HIV-1 Genomes

Retroviral recombination is thought to play an important role in the generation of immune escape and multiple drug resistance by shuffling pre-existing mutations in the viral population. Current estimates of HIV-1 recombination rates are derived from measurements within reporter gene sequences or genetically divergent HIV sequences. These measurements do not mimic the recombination occurring in vivo, between closely related genomes. Additionally, the methods used to measure recombination make a variety of assumptions about the underlying process, and often fail to account adequately for issues such as co-infection of cells or the possibility of multiple template switches between recombination sites. We have developed a HIV-1 marker system by making a small number of codon modifications in gag which allow recombination to be measured over various lengths between closely related viral genomes. We have developed statistical tools to measure recombination rates that can compensate for the possibility of multiple template switches. Our results show that when multiple template switches are ignored the error is substantial, particularly when recombination rates are high, or the genomic distance is large. We demonstrate that this system is applicable to other studies to accurately measure the recombination rate and show that recombination does not occur randomly within the HIV genome.     

Gene conversion tracts associated with crossovers in Rhizobium etli

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

Budding yeast mms4 is epistatic with rad52 and the function of Mms4 can be replaced by a bacterial Holliday junction resolvase

MMS4 of Saccharomyces cerevisiae was originally identified as the gene responsible for one of the collection of methyl methanesulfonate (MMS)-sensitive mutants, mms4. Recently it was identified as a synthetic lethal gene with an SGS1 mutation. Epistatic analyses revealed that MMS4 is involved in a pathway leading to homologous recombination requiring Rad52 or in the recombination itself, in which SGS1 is also involved. MMS sensitivity of mms4 but not sgs1, was suppressed by introducing a bacterial Holliday junction (HJ) resolvase, RusA. The frequencies of spontaneously occurring unequal sister chromatid recombination (SCR) and loss of marker in the rDNA in haploid mms4 cells and interchromosomal recombination between heteroalleles in diploid mms4 cells were essentially the same as those of wild-type cells. Although UV- and MMS-induced interchromosomal recombination was defective in sgs1 diploid cells, hyper-induction of interchromosomal recombination was observed in diploid mms4 cells, indicating that the function of Mms4 is dispensable for this type of recombination.     

The Role of DNA Repair Genes in Recombination between Repeated Sequences in Yeast

The presence of repeated sequences in the genome represents a potential source of karyotypic instability. Genetic control of recombination is thus important to preserve the integrity of the genome. To investigate the genetic control of recombination between repeated sequences, we have created a series of isogenic strains in which we could assess the role of genes involved in DNA repair in two types of recombination: direct repeat recombination and ectopic gene conversion. Naturally occurring (Ty elements) and artificially constructed repeats could be compared in the same cell population. We have found that direct repeat recombination and gene conversion have different genetic requirements. The role of the RAD51, RAD52, RAD54, RAD55, and RAD57 genes, which are involved in recombinational repair, was investigated. Based on the phenotypes of single and double mutants, these genes can be divided into three functional subgroups: one composed of RAD52, a second one composed of RAD51 and RAD54, and a third one that includes the RAD55 and RAD57 genes. Among seven genes involved in excision repair tested, only RAD1 and RAD10 played a role in the types of recombination studied. We did not detect a differential effect of any rad mutation on Ty elements as compared to artificially constructed repeats.     

Stepwise activation of the immunoglobulin mu heavy chain gene locus.

The immunoglobulin heavy chain (IgH) gene locus spans several megabases. We show that IgH activation during B-cell differentiation, as measured by histone acetylation, occurs in discrete, independently regulated domains. Initially, a 120 kb domain of germline DNA is hyperacetylated, that extends from D(FL16.1), the 5'-most D(H) gene segment, to the intergenic region between Cmu and Cdelta. Germline V(H) genes were not hyperacetylated at this stage, which accounts for D(H) to J(H) recombination occurring first during B-cell development. Subsequent activation of the V(H) locus happens in at least three differentially regulated domains: an interleukin-7-regulated domain consisting of the 5' J558 family, an intermediate domain and the 3' V(H) genes, which are hyperacetylated in response to DJ(H) recombination. These observations lead to mechanisms for two well-documented phenomena in B-cell ontogeny: the sequential rearrangement of D(H) followed by V(H) gene segments, and the preferential recombination of D(H)-proximal V(H) genes in pro-B cells. We suggest that stepwise activation may be a general mechanism by which large segments of the genome are prepared for expression.     

High-frequency homologous recombination between duplicate chromosomal immunoglobulin mu heavy-chain constant regions.

Homologous recombination was used in a previous study to correct a 2-base-pair deletion in the third constant domain (Cmu3) of the haploid chromosomal mu gene in a mutant hybridoma cell line by transfer of a pSV2neo vector bearing a subfragment of the normal Cmu region (M.D. Baker, N. Pennell, L. Bosnoyan, and M.J. Shulman, Proc. Natl. Acad. Sci. USA 85:6432-6436, 1988). In these experiments, both gene replacement and single reciprocal crossover events were found to restore normal, cytolytic 2,4,6-trinitrophenyl-specific immunoglobulin M production to the mutant cells. In the cases of single reciprocal recombination, the structure of the recombinant mu gene is such that the normal Cmu region, in its correct position 3' of the expressed 2,4,6-trinitrophenyl-specific heavy-chain variable region, is separated from the mutant Cmu region by the integrated vector sequences. I report here that homologous recombination occurs with high frequency between the duplicate Cmu regions in mitotically growing hybridoma cells. The homologous recombination events were easily detected since they generated hybridomas that were phenotypically different from the parental cells. Analysis of the recombinant cells suggests that gene conversion is the most frequent event, occurring between 60 and 73% of the time. The remaining events consisted of single reciprocal crossovers. Intrachromatid double reciprocal recombination was not detected. The high frequency of recombination, the ability to isolate and analyze the participants in the recombination reactions, and the capacity to generate specific modifications in the immunoglobulin Cmu regions by gene targeting suggest that this system will be useful for studying mammalian chromosomal homologous recombination. Moreover, the ability to specifically modify the chromosomal immunoglobulin genes by homologous recombination should facilitate studies of immunoglobulin gene regulation and expression and provide a more convenient of engineering specifically modified antibody.     

Identification of cryptic lox sites in the yeast genome by selection for Cre-mediated chromosome translocations that confer multiple drug resistance.

The Cre recombinase efficiently causes site-specific DNA recombination at loxP sites placed into the eukaryotic genome. Since the loxP site of phage P1 is 34 base-pairs in size, the natural occurrence of this exact sequence is unlikely in any eukaryotic genome. However, related sequences may exist in eukaryotic genomes that could recombine at low efficiency with an authentic loxP site. This work identifies such cryptic lox sites in the yeast genome using a positive selection procedure that allows the detection of events occurring at a frequency of less than 1 x 10(-7). The selection is based on the disruption/reconstruction of the yeast gene YGL022. Disruption of YGL022 confers multiple drug sensitivity. Recombination events at a loxP site 5' to the structural gene restore expression of YGL022 and result in a multiple drug resistant phenotype. These drug resistant mutants all display chromosomal rearrangements resulting from low-frequency Cre-mediated recombination with an endogenous cryptic lox site. Ten such sites have been found and they have been mapped physically to a number of different yeast chromosomes. Although the efficiency of Cre-mediated recombination between loxP and such endogenous sites is quite low, it may be possible to redesign recombination substrates to improve recombination efficiency. Because of the greater complexity of the human and mouse genomes compared with yeast, an analogous situation is likely to exist in these organisms. The availability of such sites would be quite useful in the development of alternative strategies for gene therapy and in the generation of transgenic animals.     

Gene stacking by recombinases

Efficient methods of stacking genes into plant genomes are needed to expedite transfer of multigenic traits to crop varieties of diverse ecosystems. Over two decades of research has identified several DNA recombinases that carryout efficient cis and trans recombination between the recombination sites artificially introduced into the plant chromosome. The specificity and efficiency of recombinases make them extremely attractive for genome engineering. In plant biotechnology, recombinases have mostly been used for removing selectable marker genes and have rarely been extended to more complex applications. The reversibility of recombination, a property of the tyrosine family of recombinases, does not lend itself to gene stacking approaches that involve rounds of transformation for integrating genes into the engineered sites. However, recent developments in the field of recombinases have overcome these challenges and paved the way for gene stacking. Some of the key advancements include the application of unidirectional recombination systems, modification of recombination sites and transgene site modifications to allow repeated site‐specific integrations into the selected site. Gene stacking is relevant to agriculturally important crops, many of which are difficult to transform; therefore, development of high‐efficiency gene stacking systems will be important for its application on agronomically important crops, and their elite varieties. Recombinases, by virtue of their specificity and efficiency in plant cells, emerge as powerful tools for a variety of applications including gene stacking.     

Hitchhiking and recombination in birds: evidence from Mhc-linked and unlinked loci in Red-winged Blackbirds (Agelaius phoeniceus)

Hitchhiking phenomena and genetic recombination have important consequences for a variety of fields for which birds are model species, yet we know virtually nothing about naturally occurring rates of recombination or the extent of linkage disequilibrium in birds. We took advantage of a previously sequenced cosmid clone from Red-winged Blackbirds (Agelaius phoeniceus) bearing a highly polymorphic Mhc class II gene, Agph-DABI, to measure the extent of linkage disequilibrium across approximately 40 kb of genomic DNA and to determine whether non-coding nucleotide diversity was elevated as a result of physical proximity to a target of balancing selection. Application of coalescent theory predicts that the hitchhiking effect is enhanced by the larger effective population size of blackbirds compared with humans, despite the presumably higher rates of recombination in birds. We surveyed sequence polymorphism at three Mhc-linked loci occurring 1.5-40 kb away from Agph-DAB1 and found that nucleotide diversity was indistinguishable from that found at three presumably unlinked, non-coding introns (beta-actin intron 2, beta-fibrinogen intron 7 and rhodopsin intron 2). Linkage disequilibrium as measured by Lewontin's D' was found only across a few hundred base pairs within any given locus, and was not detectable among any Mhc-linked loci. Estimated rates of the per site recombination rate p derived from three different analytical methods suggest that the amounts of recombination in blackbirds are up to two orders of magnitude higher than in humans, a discrepancy that cannot be explained entirely by the higher effective population size of blackbirds relative to humans. In addition, the ratio of the number of estimated recombination events per mutation frequently exceeds 1, as in Drosophila, again much higher than estimates in humans. Although the confidence limits of the blackbird estimates themselves span an order of magnitude, these data suggest that in blackbirds the hitchhiking effect for this region is negligible and may imply that the per site per individual recombination rate is high, resembling those of Drosophila more than those of humans.     

Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates.

Genetic studies of Mycobacterium tuberculosis have been greatly hampered by the inability to introduce specific chromosomal mutations. Whereas the ability to perform allelic exchanges has provided a useful method of gene disruption in other organisms, in the clinically important species of mycobacteria, such as M. tuberculosis and Mycobacterium bovis, similar approaches have thus far been unsuccessful. In this communication, we report the development of a shuttle mutagenesis strategy that involves the use of long linear recombination substrates to reproducibly obtain recombinants by allelic exchange in M. tuberculosis. Long linear recombination substrates, approximately 40 to 50 kb in length, were generated by constructing libraries in the excisable cosmid vector pYUB328. The cosmid vector could be readily excised from the recombinant cosmids by digestion with PacI, a restriction endonuclease for which there exist few, if any, sites in mycobacterial genomes. A cosmid containing the mycobacterial leuD gene was isolated, and a selectable marker conferring resistance to kanamycin was inserted into the leuD gene in the recombinant cosmid by interplasmid recombination in Escherichia coli. A long linear recombination substrate containing the insertionally mutated leuD gene was generated by PacI digestion. Electroporation of this recombination substrate containing the insertionally mutated leuD allele resulted in the generation of leucine auxotrophic mutants by homologous recombination in 6% of the kanamycin-resistant transformants for both the Erdman and H37Rv strains of M. tuberculosis. The ability to perform allelic exchanges provides an important approach for investigating the biology of this pathogen as well as developing new live-cell M. tuberculosis-based vaccines.