Effects of truncation at the non-homologous region of a family 3 beta-glucosidase from Agrobacterium tumefaciens

The function of the non-homologous region of a family 3 beta-glucosidase from Agrobacterium tumefaciens (Cbg1) was studied by analyzing the properties of mutant enzymes that have internal truncated amino acid sequences in the region. Five truncated mutants named Cbg1-d4, Cbg1-d31, Cbg1-d62, Cbg1-d89, and Cbg1-d119 having deletions of 4, 31, 62, 89, and 119 amino acid residues starting from Phe417, respectively, were expressed in Escherichia coli and purified. All the mutants exhibited beta-glucosidase activity, indicating that the non-homologous region was not essential for the activity. The truncation caused thermal instability, decrease in pK(a) of the proton donor residue (Glu616), and deficient transglycosylation activity. The thermal stability and the pK(a) of Glu616 were partially recovered with longer truncation, suggesting that the truncation perturbed the structure and that their presence in the region was not essential. The main role of the non-homologous region could be formation of a hydrophobic atmosphere at the acceptor site to make the enzyme suitable for hydrolyzing hydrophobic glucosides.     

Non-homologous synaptonemal complex formation in a heteromorphic bivalent in Keyacris scurra (Morabinae,Orthoptera)

In some populations of the grasshopper Keyacris scurra, there are many individuals heterozygous for centromere position polymorphisms. From a consideration of chiasma positions these are almost certainly due to pericentric inversions. In this species, as in other grasshoppers, the deleterious effects of chiasmata within these heterozygous regions are avoided by non-homologous, straight pairing. Reconstruction of synaptonemal complexes from two adjacent pachytene nuclei in an individual heterozygous for centromere position (telo/metacentric) on the second longest (CD) bivalent by electron microscopy of serial sections allowed the identification of all the bivalents. The centromeres were identified by characteristic densestaining material. The synaptonemal complex was found to form straight through the heteromorphic region, including both centromere positions. The pairing was clearly non-homologous at these asymmetrical centromere positions, and probably therefore, in the presumably inverted region between them. This regular non-homologous pairing explains why chiasmata never form in the heterozygous region, but does not conclusively prove that the rearrangement is an inversion rather than a centric transposition.     

RAD51 supports spontaneous non-homologous recombination in mammalian cells, but not the corresponding process induced by topoisomerase inhibitors

The RAD51 protein has been shown to participate in homologous recombination by promoting ATP-dependent homologous pairing and strand transfer reactions. In the present study, we have investigated the possible involvement of RAD51 in non-homologous recombination. We demonstrate that overexpression of CgRAD51 enhances the frequency of spontaneous non-homologous recombination in the hprt gene of Chinese hamster cells. However, the rate of non-homologous recombination induced by the topoisomerase inhibitors campothecin and etoposide was not altered by overexpression of RAD51. These results indicate that the RAD51 protein may perform a function in connection with spontaneous non-homologous recombination that is not essential to or not rate-limiting for non-homologous recombination induced by camptothecin or etoposide. We discuss the possibility that the role played by RAD51 in non-homologous recombination observed here may not be linked to non-homologous end-joining.     

Role of RNA structure in non-homologous recombination between genomic molecules of brome mosaic virus

Brome mosaic virus (BMV) is a tripartite genome, positive-sense RNA virus of plants. Previously it was demonstrated that local hybridization between BMV RNAs (RNA–RNA heteroduplex formation) efficiently promotes non-homologous RNA recombination. In addition, studies on the role of the BMV polymerase in RNA recombination suggested that the location of non-homologous crossovers depends mostly on RNA structure. As a result, a detailed analysis of a large number of non-homologous recombinants generated in the BMV-based system was undertaken. Recombination hot-spots as well as putative elements in RNA structure enhancing non-homologous crossovers and targeting them in a site-specific manner were identified. To verify these observations the recombinationally active sequence in BMV RNA3 derivative was modified. The results obtained with new RNA3 mutants suggest that the primary and secondary structure of the sequences involved in a heteroduplex formation rather than the length of heteroduplex plays the most important role in the recombination process. The presented data indicate that the sequences proximal to the heteroduplex may also affect template switching by BMV replicase. Moreover, it was shown that both short homologous sequences and a hairpin structure have to accompany a double-stranded region to target non-homologous crossovers in a site-specific manner.     

Effect of Ku86 and DNA-PKcs deficiency on non-homologous end-joining and homologous recombination using a transient transfection assay

In mammalian cells, DNA double-strand breaks are repaired by non-homologous end-joining and homologous recombination, both pathways being essential for the maintenance of genome integrity. We determined the effect of mutations in Ku86 and DNA-PK on the efficiency and the accuracy of double-strand break repair by non-homologous end-joining and homologous recombination in mammalian cells. We used an assay, based on the transient transfection of a linearized plasmid DNA, designed to simultaneously detect transfection and recombination markers. In agreement with previous results non-homologous end-joining was largely compromised in Ku86 deficient cells, and returned to normal in the Ku86-complemented isogenic cell line. In addition, analysis of DNA plasmids recovered from Ku86 mutant cells showed an increased use of microhomologies at the nonhomologous end joining junctions, and displayed a significantly higher frequency of DNA insertions compared to control cells. On the other hand, the DNA-PKcs deficient cell lines showed efficient double-strand break repair by both mechanisms.     

The mechanism of non-homologous end-joining: a synopsis of synapsis

Repair of DNA double-strand breaks (DSBs) by non-homologous end-joining (NHEJ) is required for resistance to genotoxic agents, such as ionizing radiation, but also for proper development of the vertebrate immune system. Much progress has been made in identifying the factors that are involved in this repair pathway. We are now entering the phase in which we begin to understand basic concepts of the reaction mechanism and regulation of non-homologous end-joining. This review concentrates on novel insights into damage recognition and subsequent tethering, processing and joining of DNA ends.     

Non-homologous chromosome pairing and crossover formation in haploid rice meiosis

While many studies have provided significant insight into homolog pairing during meiosis, information on non-homologous pairing is much less abundant. In the present study, fluorescence in situ hybridization (FISH) was used to investigate non-homologous pairing in haploid rice during meiosis. At pachytene, non-homologous chromosomes paired and formed synaptonemal complexes. FISH analysis data indicated that chromosome pairing could be grouped into three major types: (1) single chromosome paired fold-back as the univalent structure, (2) two non-homologous chromosomes paired as the bivalent structure, and (3) three or more non-homologous chromosomes paired as the multivalent structure. In the survey of 70 cells, 65 contained univalents, 45 contained bivalents, and 49 contained multivalent. Moreover, chromosomes 9 and 10 as well as chromosomes 11 and 12 formed non-homologous bivalents at a higher frequency than the other chromosomes. However, chiasma was always detected in the bivalent only between chromosomes 11 and 12 at diakinesis or metaphase I, indicating the pairing between these two chromosomes leads non-homologous recombination during meiosis. The synaptonemal complex formation between non-homologs was further proved by immunodetection of RCE8, PAIR2, and ZEP1. Especially, ZEP1 only loaded onto the paired chromosomes other than the un-paired chromosomes at pachytene in haploid.     

Meiotic chromosome synapsis in a haploid yeast

An extensive synaptonemal complex (SC) is found at pachytene in whole mount spread preparations of a haploid yeast, Saccharomyces cerevisiae, strain. Whereas unsynapsed axial elements are present only in a few nuclei, in others non-homologous synapsis involves virtually the whole chromosome set. This suggests that homology is not an indispensable precondition for SC formation in yeast but that chromosomes engage in non-homologous synapsis if no homologous partner is available. Recent evidence that in the sporulation deficient yeast mutants rad50 and mer1 axial elements do form but remain unsynapsed in the majority of nuclei is discussed in the light of the above findings.by D. Schweizer     

Choosing the right path: does DNA-PK help make the decision?

DNA double-strand breaks are extremely harmful lesions that can lead to genomic instability and cell death if not properly repaired. There are at least three pathways that are responsible for repairing DNA double-strand breaks in mammalian cells: non-homologous end joining, homologous recombination and alternative non-homologous end joining. Here we review each of these three pathways with an emphasis on the role of the DNA-dependent protein kinase, a critical component of the non-homologous end joining pathway, in influencing which pathway is ultimately utilized for repair.     

Illegitimate recombination as a possible mechanism of DNA-topoisomerase II induced chromosomal rearrangements

Using semi-quantitative PCR-based approach, we have shown that the breakpoint cluster region of the AML1 gene was associated with the nuclear matrix. We have demonstrated that inhibition of topoisomerase II by etoposide stimulates the appearance of histone gammaH2AX foci, an indicator for the presence of DNA double-strand breaks. Furthermore, the major part of these foci was associated with the nuclear matrix. We also visualized nuclear matrix--associated multiprotein complexes involved in topoisomerase II--induced DNA double-strand break repair. Colocalization studies have demonstrated that these complexes included the principal components of the non-homologous end joining repair system (Ku80, DNA-PKcs and DNA ligase IV). Thus, it is reasonable to suggest that the non-homologous DNA end joining is a possible mechanism of topoisomerase II--induced chromosomal rearrangements.     

Molecular mechanisms of exon shuffling: illegitimate recombination

Illegitimate recombination (IR) is a process that takes place far more often than homologous recombination and is characterized by the recombination between non-homologous or short homologous sequences. The consequences of IR frequently emerge after the introduction of DNA in cell lines because it more frequently integrates in non-homologous than in homologous regions of the host genome. As a result, unexpected truncated or elongated products may be found. By not discarding those products as transfection artifacts, but by studying how they are generated, it might elucidate a possible molecular mechanism of IR. Here we review the current literature describing different mechanisms by which non-homologous DNA recombination can be induced.     

Repair of a specific double-strand break generated within a mammalian chromosome by yeast endonuclease I-SceI.

We established a mouse Ltk- cell line that contains within its genome a herpes simplex virus thymidine kinase gene (tk) that had been disrupted by the insertion of the recognition sequence for yeast endonuclease I-SceI. The artificially introduced 18 bp I-SceI recognition sequence was likely a unique sequence in the genome of the mouse cell line. To assess whether an induced double-strand break (DSB) in the genomic tk gene would be repaired preferentially by gene targeting or non-homologous recombination, we electroporated the mouse cell line with endonuclease I-SceI alone, one of two different gene targeting constructs alone, or with I-SceI in conjunction with each of the two targeting constructs. Each targeting construct was, in principle, capable of correcting the defective genomic tk sequence via homologous recombination. tk+ colonies were recovered following electroporation of cells with I-SceI in the presence or absence of a targeting construct. Through the detection of small deletions at the I-SceI recognition sequence in the mouse genome, we present evidence that a specific DSB can be introduced into the genome of a living mammalian cell by yeast endonuclease I-SceI. We further report that a DSB in the genome of a mouse Ltk- cell is repaired preferentially by non-homologous end-joining rather than by targeted homologous recombination with an exogenous donor sequence. The potential utility of this system is discussed.     

Heterosynapsis in a heterozygous fertile boar carrier of a 3;7 translocation

Silver-stained synaptonemal complexes (SCs) in surface-spread pachytene nuclei from a boar, heterozygous for a reciprocal translocation, were analysed by electron microscopy. In such heterozygotes, cross-shaped quadrivalent configurations are expected to form in order to maximize homologous pairing. Contrary to the classical, expected cross-shaped configuration, heterosynapsis was often observed, with asymmetrical association in the lateral elements of the non-homologous partners of the quadrivalents. This heterosynapsis is assumed to be a mechanism preventing spermatocyte loss, but inducing a secondary segregational type of impairment of fertility due to foetal wastage leading to reduced prolificacy.     

A transient assay for recombination demonstrates that Arabidopsis SNM1 and XRCC3 enhance non-homologous recombination

Replacement of endogenous genes by homologous recombination is rare in plants; the majority of genetic modifications are the result of transforming DNA molecules undergoing random genomic insertion by way of non-homologous recombination. Factors that affect chromatin remodeling and DNA repair are thought to have the potential to enhance the frequency of homologous recombination in plants. Conventional tools to study the frequencies of genetic recombination often rely on stable transformation-based approaches, with these systems being rarely capable of high-throughput or combinatorial analysis. We developed a series of vectors that use chemiluminescent (LUC and REN) reporter genes to assay the relative frequency of homologous and non-homologous recombination in plants. These transient assay vectors were used to screen 14 candidate genes for their effects on recombination frequencies in Nicotiana benthamiana plants. Over-expression of Arabidopsis genes with sequence similarity to SNM1 from yeast and XRCC3 from humans enhanced the frequency of non-homologous recombination when assayed using two different donor vectors. Transient N. benthamiana leaf systems were also used in an alternative assay for preliminary measurements of homologous recombination frequencies, which were found to be enhanced by over-expression of RAD52, MIM and RAD51 from yeast, as well as CHR24 from Arabidopsis. The findings for the assays described here are in line with previous studies that analyzed recombination frequencies using stable transformation. The assays we report have revealed functions in non-homologous recombination for the Arabidopsis SNM1 and XRCC3 genes, so the suppression of these genes' expression offers a potential means to enhance the gene targeting frequency in plants. Furthermore, our findings also indicate that plant gene targeting frequencies could be enhanced by over-expression of RAD52, MIM, CHR24, and RAD51 genes.     

Rapid assessment of two major repair activities against DNA double-strand breaks in vertebrate cells

A linearized plasmid DNA, in which tandem repeats of 400bp flank the breakpoints, was transfected into vertebrate cells, and breakpoint junctions of plasmid DNA circularized in the cells were analyzed to assess the repair activities against DNA double-strand break (DSB) by non-homologous end joining and homology-directed repair (i.e., homologous recombinational repair and single-strand annealing). The circularization by non-homologous end joining repair of the breakpoints depended on the expression of DNA-PKcs, while that by homology-directed repair through the repeats depended on the length of the repeats, indicating that these two DSB repair activities can be rapidly assessed by this assay. Predominance in circularization by either non-homologous end joining or homology-directed repair differed among cells examined, and circularization was exclusively undertaken by homology-directed repair in DT40 cells known to show a high homologous recombination rate against gene-targeting vectors. Thus, this assay will be helpful in studies on mechanisms and inter-cellular variations of DSB repair.     

MgLig4, a homolog of Neurospora crassa Mus-53 (DNA ligase IV), is involved in, but not essential for, non-homologous end-joining events in Magnaporthe grisea

In many eukaryotic organisms, the non-homologous end-joining (NHEJ) system is a major pathway for the repair of DNA double-strand breaks (DSBs). DNA ligase IV is a component of the NHEJ system and is strictly required for the NHEJ system in Saccharomyces cerevisiae and in Neurospora crassa. To investigate the functions of DNA Ligase IV in Magnaporthe grisea, we generated deletion mutants of MGLIG4, which encodes a homolog of N. crassa DNA Ligase IV. Mutants (mglig4) showed no defects in asexual or sexual growth, and were fully pathogenic. Compared to the wild-type, mglig4 exhibited weak sensitivity to a DNA-damaging agent, camptothecin. In addition, the frequency of targeted-gene replacement was relatively elevated in mglig4, although this varied in a gene-dependent manner. Surprisingly, non-homologous integration of DNA was frequently observed in mglig4 transformants. Our results demonstrate that MgLig4 is involved in, but not essential for, the NHEJ system in M. grisea.     

Beauty Is in the Eye of the Beholder: Proteins Can Recognize Binding Sites of Homologous Proteins in More than One Way

Understanding the mechanisms of protein–protein interaction is a fundamental problem with many practical applications. The fact that different proteins can bind similar partners suggests that convergently evolved binding interfaces are reused in different complexes. A set of protein complexes composed of non-homologous domains interacting with homologous partners at equivalent binding sites was collected in 2006, offering an opportunity to investigate this point. We considered 433 pairs of protein–protein complexes from the ABAC database (AB and AC binary protein complexes sharing a homologous partner A) and analyzed the extent of physico-chemical similarity at the atomic and residue level at the protein–protein interface. Homologous partners of the complexes were superimposed using Multiprot, and similar atoms at the interface were quantified using a five class grouping scheme and a distance cut-off. We found that the number of interfacial atoms with similar properties is systematically lower in the non-homologous proteins than in the homologous ones. We assessed the significance of the similarity by bootstrapping the atomic properties at the interfaces. We found that the similarity of binding sites is very significant between homologous proteins, as expected, but generally insignificant between the non-homologous proteins that bind to homologous partners. Furthermore, evolutionarily conserved residues are not colocalized within the binding sites of non-homologous proteins. We could only identify a limited number of cases of structural mimicry at the interface, suggesting that this property is less generic than previously thought. Our results support the hypothesis that different proteins can interact with similar partners using alternate strategies, but do not support convergent evolution.     

One ring to bring them all—The role of Ku in mammalian non-homologous end joining

The repair of DNA double strand breaks is essential for cell survival and several conserved pathways have evolved to ensure their rapid and efficient repair. The non-homologous end joining pathway is initiated when Ku binds to the DNA break site. Ku is an abundant nuclear heterodimer of Ku70 and Ku80 with a toroidal structure that allows the protein to slide over the broken DNA end and bind with high affinity. Once locked into placed, Ku acts as a tool-belt to recruit multiple interacting proteins, forming one or more non-homologous end joining complexes that act in a regulated manner to ensure efficient repair of DNA ends. Here we review the structure and functions of Ku and the proteins with which it interacts during non-homologous end joining.     

Adaptive evolution of a melanized fungus reveals robust augmentation of radiation resistance by abrogating non-homologous end-joining

Fungi have been observed to exhibit resistance to high levels of ionizing radiation despite sharing most DNA repair mechanisms with other eukaryotes. Radioresistance, in fact, is such a common feature in fungi that it is difficult to identify species that exhibit widely different radiosensitivities, which in turn has hampered the identification of genetic elements responsible for this resistance phenotype. Due to the inherent mutagenic properties of radiation exposure, however, this can be addressed through adaptive laboratory evolution for increased ionizing radiation resistance. Here, using the black yeast Exophiala dermatitidis, we demonstrate that resistance to γ-radiation can be greatly increased through repeated rounds of irradiation and outgrowth. Moreover, we find that the small genome size of fungi situates them as a relatively simple functional genomics platform for identification of mutations associated with ionizing radiation resistance. This enabled the identification of genetic mutations in genes encoding proteins with a broad range of functions from 10 evolved strains. Specifically, we find that greatly increased resistance to γ-radiation is achieved in E. dermatitidis through disruption of the non-homologous end-joining pathway, with three individual evolutionary paths converging to abolish this DNA repair process. This result suggests that non-homologous end-joining, even in haploid cells where homologous chromosomes are not present during much of the cell cycle, is an impediment to repair of radiation-induced lesions in this organism, and that the relative levels of homologous and non-homologous repair in a given fungal species may play a major role in its radiation resistance.     

Sequence analysis, and chromosomal localization of a gene encoding a cystatin-like protein from Drosophila melanogaster.

Using polyclonal antibodies raised against a Drosophila Ca2(+)-binding protein (DCABP-23), clones were isolated from a Drosophila head cDNA library constructed in the expression vector lambda gt11. Two non-homologous clones have been isolated and are being subjected to sequence analysis. One of these clones, though not encoding DCABP-23, does encode a Drosophila cystatin-like protein. This presumed Drosophila cystatin shows homology to mammalian cystatins, chicken egg white cystatin and the rice oryzacystatin. The Drosophila cystatin has been mapped, by in situ hybridization, to region 88C on the right arm of the third chromosome.