The Effect of Nonhomologous DNA Sequences on Interplasmidic Recombination

The effect of nonhomologous DNA sequences at one or both sides of short genetic intervals on recombination within that interval was investigated, using an interplasmidic recombination system in Escherichia coli K-12. The recombining plasmids were derivatives of pBR322 and pACYC184, which share a 1330-nucleotide sequence that includes the tet gene. The genetic interval was defined by the HindIII and BamHI or BamHI and the SalI restriction endonuclease sites of this gene. The substantial differences between recombination frequencies measured within intervals bracketed or bounded on one side by major nonhomologies suggests that, in this system, strand exchange is polar and is blocked by major nonhomologies. This conclusion is substantiated by results of three-factor crosses and by structural analysis of recombination products. Results of two-factor crosses in recA genetic background and structural analysis of recombination products suggest that strand exchange occurs in the absence of a functional recA gene. "Opening" a bracketed BamHI-SalI genetic interval of the tet gene, at the SalI site, by substituting a major insertion with a short deletion, results in an increase in recombination frequency, within this genetic interval, which is greater than expected on the basis of the ratio of the length of homology on the two sides of the SalI site. This observation suggests that a genetic element that may affect rate of recombination initiation, polarity of strand exchange, template specificity in mismatch repair or more than one of these events may be present on the outer side of the SalI site of the tet gene.     

Genetic Polymorphism and Recombination in the Subtelomeric Region of Chromosome 14q

Subtelomeric regions of human chromosomes are the sites of increased meiotic recombination and have a male-to-female recombination ratio that is higher than elsewhere in the genome. We isolated two novel, polymorphic CA repeat markers from the distal part of the immunoglobulin heavy chain gene cluster, approximately 90 and 200 kb from the telomere of chromosome 14q. The 14q telomere was unambiguously located by physical mapping of telomeric YACs andBal31 exonuclease digestion of genomic DNA. We then constructed haplotypes using genotype data from these markers and data from sCAW1 (D14S826) for use as a highly polymorphic genetic marker. Linkage analysis using the 40 pedigree CEPH reference panel and genotype data from these and other loci physically mapped to the terminal 1.5 Mb of chromosome 14q revealed an apparent increase in meiotic recombination within this region, relative to the average rate for the genome. Further, we found that recombination was higher in females than in males, indicating that the subtelomeric region of 14q differs from other human subtelomeric regions.     

Characterization of Telomeric Oligonucleotides: Fidelity of Repetitive Nucleotide Sequences§

Abstract

To clarify the reasons for the high fidelity of repetitive telomeric sequences, a series of d(TTXGGG)₄ (X: A, G, C, and T) were synthesized and characterized by UV absorption, CD, chemical modification, and resistance to nucleases. d(TTCGGG)₄, which is lacking in nature, has similar structural stability and resistance to nucleases, compared with d(TTAGGG)₄, which is widely present at the telomeres of many organisms. d(TTGGGG)₄ is the most stable as a result of formation of the four G-quartet layers in the presence of potassium ion.

     

Repair and Reconstruction of Telomeric and Subtelomeric Regions and Genesis of New Telomeres: Implications for Chromosome Evolution

DNA damage repair within telomeres are suppressed to maintain the integrity of linear chromosomes, but the accidental activation of repairs can lead to genome instability. This review develops the concept that mechanisms to repair DNA damage in telomeres contribute to genetic variability and karyotype evolution, rather than catastrophe. Spontaneous breaks in telomeres can be repaired by telomerase, but in some cases DNA repair pathways are activated, and can cause chromosomal rearrangements or fusions. The resultant changes can also affect subtelomeric regions that are adjacent to telomeres. Subtelomeres are actively involved in such chromosomal changes, and are therefore the most variable regions in the genome. The case of Caenorhabditis elegans in the context of changes of subtelomeric structures revealed by long-read sequencing is also discussed. Theoretical and methodological issues covered in this review will help to explore the mechanism of chromosome evolution by reconstruction of chromosomal ends in nature.     

The Evolution of Restricted Recombination and the Accumulation of Repeated DNA Sequences

We suggest hypotheses to account for two major features of chromosomal organization in higher eukaryotes. The first of these is the general restriction of crossing over in the neighborhood of centromeres and telomeres. We propose that this is a consequence of selection for reduced rates of unequal exchange between repeated DNA sequences for which the copy number is subject to stabilizing selection: microtubule binding sites, in the case of centromeres, and the short repeated sequences needed for terminal replication of a linear DNA molecule, in the case of telomeres. An association between proximal crossing over and nondisjunction would also favor the restriction of crossing over near the centromere. The second feature is the association between highly repeated DNA sequences of no obvious functional significance and regions of restricted crossing over. We show that highly repeated sequences are likely to persist longest (over evolutionary time) when crossing over is infrequent. This is because unequal exchange among repeated sequences generates single copy sequences, and a population that becomes fixed for a single copy sequence by drift remains in this state indefinitely (in the absence of gene amplification processes). Increased rates of exchange thus speed up the process of stochastic loss of repeated sequences.     

Fine-Structure Mapping of Meiosis-Specific Double-Strand DNA Breaks at a Recombination Hotspot Associated with an Insertion of Telomeric Sequences Upstream of the His4 Locus in Yeast

Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand DNA breaks (DSBs). Using two approaches, we mapped the position of DSBs associated with a recombination hotspot created by insertion of telomeric sequences into the region upstream of HIS4. We found that the breaks have no obvious sequence specificity and localize to a region of ~50 bp adjacent to the telomeric insertion. By mapping the breaks and by studies of the exonuclease III sensitivity of the broken ends, we conclude that most of the broken DNA molecules have blunt ends with 3'-hydroxyl groups.     

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.     

The Schizosaccharomyces pombe JmjC-Protein,Msc1, Prevents H2A.Z Localization in Centromeric and Subtelomeric Chromatin Domains

Eukaryotic genomes are repetitively packaged into chromatin by nucleosomes, however they are regulated by the differences between nucleosomes, which establish various chromatin states. Local chromatin cues direct the inheritance and propagation of chromatin status via self-reinforcing epigenetic mechanisms. Replication-independent histone exchange could potentially perturb chromatin status if histone exchange chaperones, such as Swr1C, loaded histone variants into wrong sites. Here we show that in Schizosaccharomyces pombe, like Saccharomyces cerevisiae, Swr1C is required for loading H2A.Z into specific sites, including the promoters of lowly expressed genes. However S. pombe Swr1C has an extra subunit, Msc1, which is a JumonjiC-domain protein of the Lid/Jarid1 family. Deletion of Msc1 did not disrupt the S. pombe Swr1C or its ability to bind and load H2A.Z into euchromatin, however H2A.Z was ectopically found in the inner centromere and in subtelomeric chromatin. Normally this subtelomeric region not only lacks H2A.Z but also shows uniformly lower levels of H3K4me2, H4K5, and K12 acetylation than euchromatin and disproportionately contains the most lowly expressed genes during vegetative growth, including many meiotic-specific genes. Genes within and adjacent to subtelomeric chromatin become overexpressed in the absence of either Msc1, Swr1, or paradoxically H2A.Z itself. We also show that H2A.Z is N-terminally acetylated before, and lysine acetylated after, loading into chromatin and that it physically associates with the Nap1 histone chaperone. However, we find a negative correlation between the genomic distributions of H2A.Z and Nap1/Hrp1/Hrp3, suggesting that the Nap1 chaperones remove H2A.Z from chromatin. These data describe H2A.Z action in S. pombe and identify a new mode of chromatin surveillance and maintenance based on negative regulation of histone variant misincorporation.     

Physical Mapping of Human 7q and 14q Subtelomeric DNA Sequences in the Great Apes

Phylogenetic divergence of the members of the Pongidae familyhas been based on genetic evidence. The terminal repeat array(T₂AG₃) has lately been considered as an additional basis toanalyze genomes of highly related species. The recent isolationof subtelomeric DNA probes specific for human (HSA) chromosomes7q and 14q has prompted us to cross-hybridize them to the chromosomesof the chimpanzee (PTR), gorilla (GGO) and orangutan (PPY) tosearch for its equivalent locations in the great ape species.Both probes hybridized to the equivalent telomeric sites ofthe long (q) arms of all three great ape species. Hybridizationsignals to the 7q subtelomeric DNA sequence probe were observedat the telomeres of HSA 7q, PTR 6q, GGO 6q and PPY 10q, whilehybridization signals to the 14q subtelomeric DNA sequence probewere observed at the telomeres of HSA 14q, PTR 15q, GGO 18qand PPY 15q. No hybridization signals to the chromosome 7-specificalpha satellite DNA probe on the centromeric regions of theape chromosomes were observed. Our observations demonstratesequence homology of the subtelomeric repeat families D7S427and D14S308 in the ape chromosomes. An analogous number of subtelomericrepeat units exists in these chromosomes and has been preservedthrough the course of differentiation of the hominoid species.Our investigation also suggests a difference in the number ofalpha satellite DNA repeat units in the equivalent ape chromosomes,possibly derived from interchromosomal transfers and subsequentamplification of ancestral alpha satellite sequences.     

The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo

In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Δ cells. Hence, we conclude that CEB1 instability in pif1Δ cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences.     

The Efficiency of Meiotic Recombination between Dispersed Sequences in Saccharomyces Cerevisiae Depends upon Their Chromosomal Location

To examine constraints imposed on meiotic recombination by homologue pairing, we measured the frequency of recombination between mutant alleles of the ARG4 gene contained in pBR322-based inserts. Inserts were located at identical loci on homologues (allelic recombination) or at different loci on either homologous or heterologous chromosomes (ectopic recombination). Ectopic recombination between interstitially located inserts on heterologous chromosomes had an efficiency of 6-12% compared to allelic recombination. By contrast, ectopic recombination between interstitial inserts located on homologues had relative efficiencies of 47-99%. These findings suggest that when meiotic ectopic recombination occurs, homologous chromosomes are already colocalized. The efficiency of ectopic recombination between inserts on homologues decreased as the physical distance between insert sites was increased. This result is consistent with the suggestion that during meiotic recombination, homologues are not only close to each other, but also are aligned end to end. Finally, the efficiency of ectopic recombination between inserts near telomeres (within 16 kb) was significantly greater than that observed with inserts >50 kb from the nearest telomere. Thus, at the time of recombination, there may be a special relationship between the ends of chromosomes not shared with interstitial regions.     

Red/ET 同源重组介导细菌人工染色体的快速修饰

随着基因组测序工程的实施与完成,如何对包含完整基因信息的特定细菌人工染色体 (BAC) 进行有目的修饰,已成为功能基因组学研究的一个重要环节 . 应用新近优化的 Red/ET 同源重组技术对目标 BAC 进行修饰,以 pSC101-BAD-gbaA 为依托质粒,采用 rpsL-neo 为正 / 反向筛选系统,可以快速、高效地对 BAC 进行剪切、插入、替换等操作,其中能够进行抗性筛选的一步 BAC 修饰只需一周时间,以插入非抗性标记基因 Cre 为代表的两步 BAC 修饰在两周内即可完成 . 通过阿拉伯多糖诱导调控和简单地变化培养温度,能使 pSC101-BAD-gbaA 依托质粒在发挥完 Red/ET 同源重组作用后自然消失,最终获得完整而纯净的修饰后 BAC ,为加快功能基因组学研究提供了一个可靠的实验平台 .     

The Detection of Inherent Homologous Recombination Between Repeat Sequences in H. pylori 26695 by the PCR-Based Method

Helicobacter pylori infects more than half of the world’s population, making it the most widespread infection of bacteria. It has high genetic diversity and has been considered as one of the most variable bacterial species. In the present study, a PCR-based method was used to detect the presence and the relative frequency of homologous recombination between repeat sequences (>500 bp) in H. pylori 26695. All the recombinant structures have been confirmed by sequencing. The inversion generated between inverted repeats showed distinct features from the recombination for duplication or deletion between direct repeats. Meanwhile, we gave the mathematic reasoning of a general formula for the calculation of relative recombination frequency and indicated the conditions for its application. This formula could be extensively applied to detect the frequency of homologous recombination, site-specific recombination, and other types of predictable recombination. Our results should be helpful for better understanding the genome evolution and adaptation of bacteria.     

Mechanisms of Recombination between Diverged Sequences in Wild-Type and BLM-Deficient Mouse and Human Cells

Double-strand breaks (DSBs) are particularly deleterious DNA lesions for which cells have developed multiple mechanisms of repair. One major mechanism of DSB repair in mammalian cells is homologous recombination (HR), whereby a homologous donor sequence is used as a template for repair. For this reason, HR repair of DSBs is also being exploited for gene modification in possible therapeutic approaches. HR is sensitive to sequence divergence, such that the cell has developed ways to suppress recombination between diverged (“homeologous”) sequences. In this report, we have examined several aspects of HR between homeologous sequences in mouse and human cells. We found that gene conversion tracts are similar for mouse and human cells and are generally ≤100 bp, even in Msh2^−/− cells which fail to suppress homeologous recombination. Gene conversion tracts are mostly unidirectional, with no observed mutations. Additionally, no alterations were observed in the donor sequences. While both mouse and human cells suppress homeologous recombination, the suppression is substantially less in the transformed human cells, despite similarities in the gene conversion tracts. BLM-deficient mouse and human cells suppress homeologous recombination to a similar extent as wild-type cells, unlike Sgs1-deficient Saccharomyces cerevisiae.The ability of a cell to repair DNA damage is integral to maintaining genome integrity. One common type of damage that is particularly detrimental is a double-strand break (DSB), where both strands of DNA are broken. If not accurately repaired, DSBs can lead to cell death, chromosomal rearrangements, and loss of genetic material (reviewed in references 14 and 19). One mechanism of DSB repair is homologous recombination (HR), in which an unbroken homologous sequence, the donor of genetic information, is used as a template for repair of the broken sequence, the recipient of genetic information. HR intermediates possess heteroduplex DNA (hDNA), where one strand of DNA is derived from the donor sequence, and the second strand is derived from the recipient sequence. Mismatches in hDNA are substrates of the mismatch repair machinery (MMR) (reviewed in reference 38), leading to gene conversion. HR is the preferred repair pathway of DSBs in Saccharomyces cerevisiae (reviewed in references 42 and 46), plays an important role in repair of DSBs in Drosophila (1, 32), and is a major repair pathway of DSBs that occur during S/G₂ in mammalian cells (33, 54).Two pathways appear to predominate for the repair of DSBs by HR, both of which can give rise to noncrossover products, which predominate in mitotic mammalian cells (Fig. ​(Fig.1)1) (29, 52, 60). In the DSB repair model proposed by Szostak et al. (61), double Holliday junctions are resolved to result in recombinant products (Fig. ​(Fig.1A).1A). More recent evidence suggests the existence of an alternative pathway, termed synthesis-dependent strand annealing (SDSA) (Fig. ​(Fig.1B)1B) (20, 40, 42, 52). One difference between these two pathways is that the DSB repair model requires capture of both DNA ends (Fig. ​(Fig.1A),1A), which can lead to bidirectional gene conversion tracts. In contrast, SDSA can involve only one end of the broken DNA followed by dissociation (Fig. ​(Fig.1B),1B), resulting in predominantly unidirectional gene conversion tracts. Another difference is that the donor sequence can be altered during DSB repair while it typically remains unchanged after SDSA.Open in a separate windowFIG. 1.Models for noncrossover gene conversion resulting from DSB repair. DSB repair is initiated by resection of the DNA ends (black; strand directionality is designated a 3′ “tail”). The resected 3′ overhang invades the homologous donor template (gray), forming hDNA at the site of invasion (i), which acts as a primer/template for repair synthesis (gray dotted line). (A) In the canonical DSB repair (DSBR) model, the second strand of the DSB is captured, resulting in another stretch of hDNA (ii) and repair synthesis, to form a double Holliday junction. Depending on how the double Holliday junction is cleaved (arrowheads), resolution can result in a crossover (data not shown) or a noncrossover, as shown. (B) In SDSA, the newly synthesized strand dissociates from the D-loop and anneals to the other DNA end to form another stretch of hDNA (iii). Repair synthesis and ligation result in a noncrossover product. While one-end invasion is illustrated for the SDSA model, it is possible for both DNA ends to invade, resulting in gene conversion on both sides of the DSB (data not shown). In both models, hDNA formed by the newly synthesized strands can be repaired by MMR, resulting in gene conversion of markers (data not shown).HR repair is sensitive to differences between the recombining sequences, and cells have developed ways to suppress recombination between diverged sequences. This suppression of “homeologous” recombination reduces HR both between diverged repeats and with foreign DNA. Suppression of homeologous recombination is conserved across species and requires the MMR machinery (7, 10, 11, 49, 56). For example, MSH2 dramatically reduces both gene targeting (12) and DSB-induced HR (15) between sequences with >1% divergence in murine embryonic stem (ES) cells.Another protein that has been proposed to suppress homeologous recombination is Sgs1, the budding yeast RecQ helicase, as sequence divergence has little effect on recombination frequencies in Sgs1 mutants (39, 59). Sgs1 mutants have other phenotypes as well; for example, they demonstrate a hyperrecombination phenotype associated with spontaneous repair (22, 65, 68). The mammalian homolog of Sgs1 is BLM, mutants of which also have a hyperrecombination phenotype, as evidenced by a high frequency of sister-chromatid exchange (SCE) in both human and mouse cells (18, 24, 34, 69). Evidence suggests that Drosophila BLM, like Sgs1, has a role in the suppression of homeologous recombination (30) although mammalian BLM has not been tested in this regard. Supporting a possible role for BLM in suppressing homeologous recombination is the observation that BLM associates with MMR factors in a large protein complex (64; reviewed in reference 21), and BLM directly interacts with two components of the MMR machinery, MLH1 (45) and MSH6 (44), which, like MSH2, is known to suppress homeologous recombination (13).To gain more insight into mammalian HR mechanisms, as well as factors that control recombination between homeologous sequences, we examined recombination between homologous and homeologous sequences in both murine and human cells. By taking advantage of multiple, single base pair polymorphisms distributed along the donor in gene conversion substrates, we examined both the nature of gene conversion tracts and the fate of the donor sequence. Unidirectional tracts with a bias in conversion to one side of the DSB predominated in both mouse and human cells, supporting an SDSA mechanism of HR. Moreover, the donor remained unaltered after HR. Interestingly, while transformed human cells suppressed homeologous recombination, the degree of suppression was less than that observed in mouse cells. For either cell type, BLM deficiency did not alter this suppression, unlike what is observed in yeast Sgs1 mutants. Either other RecQ helicase family members play a role in the suppression of homeologous recombination, or mammalian RecQ helicases do not play a role in this process.