Analysis of receptor binding displacement curves by a nonhomologous ligand, on the basis of an equivalent competition principle

An exact method for the analysis of receptor-ligand binding data when labeled bound ligand is displaced by a nonhomologous ligand with a different dissociation constant is described. The present method, which is based on an equivalent competition principle for the homologous and the nonhomologous ligand, converts displacement curves into a linear form and is also applicable to situations in which free concentrations of ligand are significantly smaller than the added concentrations as a result of ligand binding. It is shown that the dissociation constant of the nonhomologous ligand is given directly by the concentration of this nonhomologous ligand added and the free concentration of unlabeled homologous ligand required to give the same level of displacement of labeled bound ligand. On the basis of these displacement characteristics, all binding parameters for receptor interaction of the nonhomologous ligand can be obtained and expressed, for example, in a Scatchard plot. The present method, which is referred to as the equivalent competition method, is also evaluated in this study with respect to the effects of nonspecific ligand binding and the presence of multiple receptor classes.     

Removal of one nonhomologous DNA end during gene conversion by a RAD1- and MSH2-independent pathway

Repair of a double-strand break (DSB) by homologous recombination depends on the invasion of a 3'-ended strand into an intact template sequence to initiate new DNA synthesis. When the end of the invading DNA is not homologous to the donor, the nonhomologous sequences must be removed before new synthesis can begin. In Saccharomyces cerevisiae, the removal of these ends depends on both the nucleotide excision repair endonuclease Rad1p/Rad10p and the mismatch repair proteins Msh2p/Msh3p. In rad1 or msh2 mutants, when both ends of the DSB have nonhomologous ends, repair is reduced approximately 90-fold compared to a plasmid with perfect ends; however, with only one nonhomologous end, repair is reduced on average only 5-fold. These results suggest that yeast has an alternative, but less efficient, way to remove a nonhomologous tail from the second end participating in gene conversion. When the removal of one nonhomologous end is impaired in rad1 and msh2 mutants, there is also a 1-hr delay in the appearance of crossover products of gene conversion, compared to noncrossovers. We interpret these results in terms of the formation and resolution of alternative intermediates of a synthesis-dependent strand annealing mechanism.     

Activation of a system for the joining of nonhomologous DNA ends during Xenopus egg maturation.

Mature Xenopus laevis eggs provide an elementary reaction system of illegitimate recombination which efficiently joins nonhomologous DNA ends (P. Pfeiffer and W. Vielmetter, Nucleic Acids Res. 16:907-924, 1988). Here we show that stage VI oocytes, known to express a system for homologous recombination (D. Carroll, Proc. Natl. Acad. Sci. USA 80:6902-6906, 1983), are completely devoid of this joining system. Nonhomologous DNA end-to-end joining, however, attains full activity only at an extremely late stage of egg maturation. Cycloheximide inhibition patterns indicate that nonhomologous joining activity is regulated at the G2 restriction point of the cell cycle. Implications of homologous and nonhomologous recombination activities during egg maturation are discussed.     

Nonhomologous recombination in the parvovirus chromosome: role for a CTATTTCT motif.

The mechanism of nonhomologous recombination in murine cells infected with the parvovirus minute virus of mice (MVM) has been investigated by analysis of DNA sequences at recombination junctions in naturally occurring deletion variants of the virus. We report here that nonhomologous recombination in the MVM chromosome is characterized by short homologies, by insertion at recombination junctions of foreign DNA sequences that are enriched for preferred eucaryotic topoisomerase I cleavage sites, and by an association with a common DNA sequence motif of the type 5'-CTATTTCT-3'. Additional analyses of broken MVM chromosomes provided evidence for specific enzymatic cleavage within 5'-CTTATC-3' and 5'-CTATTC-3' sequences. The results indicate that the 5'-CTATTTCT-3' motif is an important genetic element for nonhomologous recombination in the parvovirus chromosome.     

Polymerase mu is a DNA-directed DNA/RNA polymerase

DNA polymerases are defined as such because they use deoxynucleotides instead of ribonucleotides with high specificity. We show here that polymerase mu (pol mu), implicated in the nonhomologous end-joining pathway for repair of DNA double-strand breaks, incorporates both ribonucleotides and deoxynucleotides in a template-directed manner. pol mu has an approximately 1,000-fold-reduced ability to discriminate against ribonucleotides compared to that of the related pol beta, although pol mu's substrate specificity is similar to that of pol beta in most other respects. Moreover, pol mu more frequently incorporates ribonucleotides when presented with nucleotide concentrations that approximate cellular pools. We therefore addressed the impact of ribonucleotide incorporation on the activities of factors required for double-strand break repair by nonhomologous end joining. We determined that the ligase required for this pathway readily joined strand breaks with terminal ribonucleotides. Most significantly, pol mu frequently introduced ribonucleotides into the repair junctions of an in vitro nonhomologous end-joining reaction, an activity that would be expected to have important consequences in the context of cellular double-strand break repair.     

The origin of a "zebra" chromosome in wheat suggests nonhomologous recombination as a novel mechanism for new chromosome evolution and step changes in chromosome number

An alloplasmic wheat line, TA5536, with the "zebra" chromosome z5A was isolated from an Elymus trachycaulus/Triticum aestivum backcross derivative. This chromosome was named "zebra" because of its striped genomic in situ hybridization pattern. Its origin was traced to nonhomologous chromosome 5A of wheat and 1H(t) of Elymus; four chromatin segments were derived from chromosome 1H(t) and five chromatin segments including the centromere from 5A. In this study, our objective was to determine the mechanism of origin of chromosome z5A, whether by nonhomologous recombination or by multiple translocation events. Different crossing schemes were used to recover recombinants containing various Elymus chromatin segments of the z5A chromosome. In addition, one z5AL telocentric chromosome and three z5AL isochromosomes were recovered. The dissection of the Elymus segments into different stocks allowed us to determine the chromosomal origin of the different chromosome fragments on the basis of the order of the RFLP markers employed and suggested that the zebra chromosome originated from nonhomologous recombination. We present a model of possible mechanism(s) of chromosome evolution and step changes in chromosome number applicable to a wide range of organisms.     

Effect of limited homology on gene conversion in a Saccharomyces cerevisiae plasmid recombination system.

Plasmids containing heteroallelic copies of the Saccharomyces cerevisiae HIS3 gene undergo intramolecular gene conversion in mitotically dividing S. cerevisiae cells. We have used this plasmid system to determine the minimum amount of homology required for gene conversion, to examine how conversion tract lengths are affected by limited homology, and to analyze the role of flanking DNA sequences on the pattern of exchange. Plasmids with homologous sequences greater than 2 kilobases have mitotic exchange rates as high as 2 x 10(-3) events per cell per generation. As the homology is reduced, the exchange rate decreases dramatically. A plasmid with 26 base pairs (bp) of homology undergoes gene conversion at a rate of approximately 1 x 10(-10) events per cell per generation. These studies have also shown that an 8-bp insertion mutation 13 bp from a border between homologous and nonhomologous sequences undergoes conversion, but that a similar 8-bp insertion 5 bp from a border does not. Examination of independent conversion events which occurred in plasmids with heteroallelic copies of the HIS3 gene shows that markers within 280 bp of a border between homologous and nonhomologous sequences undergo conversion less frequently than the same markers within a more extensive homologous sequence. Thus, proximity to a border between homologous and nonhomologous sequences shortens the conversion tract length.     

Quantitation of a simian virus 40 nonhomologous recombination pathway.

We describe an infectious-center in situ plaque hybridization procedure which quantitates simian virus 40 (SV40) nonhomologous recombination in terms of the number of recombinant-producing cells in the DNA transfected cell population. Using this assay to measure the efficiency of recombination with SV40 DNA in permissive monkey BSC-1 cells, we found that: (i) over a range of DNA concentrations, polyomavirus DNA (which is partially homologous to SV40 DNA) cannot be distinguished from nonhomologous phi X174 RF1 DNA with respect to its ability to recombine with SV40 DNA; (ii) at defined DNA concentrations, polyomavirus and phi X174 RF1 DNA compete with each other for recombination with SV40 DNA; (iii) virtually all segments of the phi X174 genome recombine, apparently at random, with SV40 DNA; (iv) the frequency of recombinant-producing cells, among the successfully transfected (virion-producing) cells, depends upon the input SV40 DNA concentration in the transfection solution; and (v) replication-defective SV40 mutant DNAs compete with wild-type SV40 DNA for recombination with phi X174 RF1 DNA. From these observations, we conclude that the efficiency of recombination with SV40, in the system under study, is unaffected by nucleotide sequence homology and that a limiting stage in the recombination pathway occurs before SV40 DNA replication. Comparison of the dependency of recombination on initial SV40 DNA concentration with the dependency on initial phi X174 RF1 DNA concentration indicates that SV40 DNA sequences are a controlling factor in the nonhomologous recombination pathway.     

Involvement of the nonhomologous region of subunit A of the yeast V-ATPase in coupling and in vivo dissociation

The catalytic nucleotide binding subunit (subunit A) of the vacuolar proton-translocating ATPase (or V-ATPase) is homologous to the beta-subunit of the F-ATPase but contains a 90-amino acid insert not present in the beta-subunit, termed the nonhomologous region. We previously demonstrated that mutations in this region lead to changes in coupling of proton transport and ATPase activity and to inhibition of in vivo dissociation of the V-ATPase complex, an important regulatory mechanism (Shao, E., Nishi T., Kawasaki-Nishi, S., and Forgac, M. (2003) J. Biol. Chem. 278, 12985-12991). Measurement of the ATP dependence of coupling for the wild type and mutant proteins demonstrates that the coupling differences are observed at ATP concentrations up to 1 mm. A decrease in coupling efficiency is observed at higher ATP concentrations for the wild type and mutant V-ATPases. Immunoprecipitation of an epitope-tagged nonhomologous region from cell lysates indicates that this region is able to bind to the integral V0 domain in the absence of the remainder of the A subunit, an interaction confirmed by immunoprecipitation of V0. Interaction between the nonhomologous region and V0 is reduced upon incubation of cells in the absence of glucose, suggesting that the nonhomologous region may act as a trigger to activate in vivo dissociation. Immunoprecipitation suggests that the epitope tag on the nonhomologous region becomes less accessible upon glucose withdrawal, possibly due to binding to another cellular target. In vivo dissociation of the V-ATPase in response to glucose removal is also blocked by chloroquine, a weak base that neutralizes the acidic pH of the vacuole. The results suggest that the dependence of in vivo dissociation of the V-ATPase on catalytic activity may be due to neutralization of the yeast vacuole, which in turn blocks glucose-dependent dissociation.     

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.     

Role of terminal nonhomologous domains in initiation of human red cell spectrin dimerization

Human erythrocyte spectrin is an antiparallel heterodimer comprised of a 280 kDa alpha subunit and a 246 kDa beta subunit which further associates into tetramers in the red cell membrane cytoskeleton. Lateral association of the flexible rodlike monomers involves a multiple-step process that is initiated by a high affinity association near the actin-binding end of the molecule (dimer nucleation site). In this study, recombinant alpha and beta proteins comprising two or four "spectrin type" motifs with and without adjacent, terminal nonhomologous domains were evaluated for their relative contributions to dimer initiation, and the thermodynamic properties of these heterodimer complexes were measured. Sedimentation equilibrium studies showed that in the absence of the heterologous subunit, individual recombinant proteins formed weak homodimers (K(d) > 0.3 mM). When 2-motif (alpha20-21 and beta1-2) and 4-motif (alpha18-21 and beta1-4) recombinants lacking the terminal nonhomologous domains were paired with the complementary protein, high affinity heterodimers were formed in sedimentation equilibrium analysis. Both the alpha20-21/beta1-2 complex and the alpha20-21EF/betaABD1-2 complex showed stoichiometric binding with similar binding affinities (K(d) approximately 10 nM) using isothermal titration calorimetry. The alpha20-21/beta1-2 complex showed an enthalpy of -10 kcal/mol, while the alpha20-21EF/betaABD1-2 complex showed an enthalpy of -13 kcal/mol. Pull-down assays using alpha spectrin GST fusion proteins showed strong associations between all heterodimer complexes in physiological buffer, but all heterodimer complexes were destabilized by the presence of Triton X-100 and other detergents. Complexes lacking the nonhomologous domains were destabilized to a greater extent than complexes that included the nonhomologous domains. The detergent effect appears to be responsible for the apparent essential role of the nonhomologous domains in prior reports. Taken together, our results indicate that the terminal nonhomologous domains do not contribute to dimer initiation nor are they required for formation of high affinity spectrin heterodimers in physiological buffers.     

Changing the antigen binding specificity by single point mutations of an anti-p24 (HIV-1) antibody

The murine mAb CB4-1 raised against p24 (HIV-1) recognizes a linear epitope of the HIV-1 capsid protein. Additionally, CB4-1 exhibits cross-reactive binding to epitope-homologous peptides and polyspecific reactions to epitope nonhomologous peptides. Crystal structures demonstrate that the epitope peptide (e-pep) and the nonhomologous peptides adopt different conformations within the binding region of CB4-1. Site-directed mutagenesis of the fragment variable (Fv) region was performed using a single-chain (sc)Fv construct of CB4-1 to analyze binding contributions of single amino acid side chains toward the e-pep and toward one epitope nonhomologous peptide. The mutations of Ab amino acid side chains, which are in direct contact with the Ag, show opposite influences on the binding of the two peptides. Whereas the affinity of the e-pep to the CB4-1 scFv mutant heavy chain variable region Tyr(32)Ala is decreased 250-fold, the binding of the nonhomologous peptide remains unchanged. In contrast, the mutation light chain variable region Phe(94)Ala reduces the affinity of the nonhomologous peptide 10-fold more than it does for the e-pep. Thus, substantial changes in the specificity can be observed by single amino acid exchanges. Further characterization of the scFv mutants by substitutional analysis of the peptides demonstrates that the effect of a mutation is not restricted to contact residues. This method also reveals an inverse compensatory amino acid exchange for the nonhomologous peptide which increases the affinity to the scFv mutant light chain variable region Phe(94)Ala up to the level of the e-pep affinity to the wild-type scFv.     

GDF6, a novel locus for a spectrum of ocular developmental anomalies

Colobomata represent visually impairing ocular closure defects that are associated with a diverse range of developmental anomalies. Characterization of a chromosome 8q21.2-q22.1 segmental deletion in a patient with chorioretinal coloboma revealed elements of nonallelic homologous recombination and nonhomologous end joining. This genomic architecture extends the range of chromosomal rearrangements associated with human disease and indicates that a broader spectrum of human chromosomal rearrangements may use coupled homologous and nonhomologous mechanisms. We also demonstrate that the segmental deletion encompasses GDF6, encoding a member of the bone-morphogenetic protein family, and that inhibition of gdf6a in a model organism accurately recapitulates the proband's phenotype. The spectrum of disorders generated by morpholino inhibition and the more severe defects (microphthalmia and anophthalmia) observed at higher doses illustrate the key role of GDF6 in ocular development. These results underscore the value of integrated clinical and molecular investigation of patients with chromosomal anomalies.     

Analysis of DNA double-strand break repair pathways in mice

During the last years significant new insights have been gained into the mechanism and biological relevance of DNA double-strand break (DSB) repair in relation to genome stability. DSBs are a highly toxic DNA lesion, because they can lead to chromosome fragmentation, loss and translocations, eventually resulting in cancer. DSBs can be induced by cellular processes such as V(D)J recombination or DNA replication. They can also be introduced by exogenous agents DNA damaging agents such as ionizing radiation or mitomycin C. During evolution several pathways have evolved for the repair of these DSBs. The most important DSB repair mechanisms in mammalian cells are nonhomologous end-joining and homologous recombination. By using an undamaged repair template, homologous recombination ensures accurate DSB repair, whereas the untemplated nonhomologous end-joining pathway does not. Although both pathways are active in mammals, the relative contribution of the two repair pathways to genome stability differs in the different cell types. Given the potential differences in repair fidelity, it is of interest to determine the relative contribution of homologous recombination and nonhomologous end-joining to DSB repair. In this review, we focus on the biological relevance of DSB repair in mammalian cells and the potential overlap between nonhomologous end-joining and homologous recombination in different tissues.     

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

Using a semiquantitative PCR-based approach, a breakpoint cluster region of AML1 was associated with the nuclear matrix. Inhibition of topoisomerase II (topoII) by etoposide stimulated the appearance of histone γH2AX foci, indicative of DNA double-strand breaks (DSBs). The majority of these foci were associated with the nuclear matrix. Nuclear matrix-associated multiprotein complexes involved in topoII-induced DNA DSB repair were visualized. Colocalization studies demonstrated that these complexes included the main components of the nonhomologous end joining repair system (Ku80, DNA-PKcs, and DNA ligase IV). Thus, it was suggested that nonhomologous DNA end joining is a possible mechanism of topoII-induced chromosomal rear-rangements.     

RNA recombination in brome mosaic virus, a model plus strand RNA virus.

Studies on the molecular mechanism of genetic recombination in RNA viruses have progressed at the time when experimental systems of efficient recombination crossovers were established. The system of brome mosaic virus (BMV) represents one of the most useful and most advanced tools for investigation of the molecular aspects of the mechanism of RNA-RNA recombination events. By using engineered BMV RNA components, the occurrence of both homologous and nonhomologous crosses were demonstrated among the segments of the BMV RNA genome. Studies show that the two types of crossovers require different RNA signal sequences and that both types depend upon the participation of BMV replicase proteins. Mutations in the two BMV-encoded replicase polypeptides (proteins 1a and 2a) reveal that their different regions participate in homologous and in nonhomologous crossovers. Based on all these data, it is most likely that homologous and nonhomologous recombinant crosses do occur via two different types of template switching events (copy-choice mechanism) where viral replicase complex changes RNA templates during viral RNA replication at distinct signal sequences. In this review we discuss various aspects of the mechanism of RNA recombination in BMV and we emphasize future projections of this research.     

Effect of <Emphasis Type="Italic">rad50</Emphasis> mutation on illegitimate recombination in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Genes in the RAD52 epistasis group are involved in repairing DNA double-stranded breaks via homologous recombination. We have previously shown that RAD50 is involved in mitotic nonhomologous integration but not in homologous integration. However, the role of Rad50 in nonhomologous integration has not previously been examined. In the current work, we report that the rad50∆ mutation caused a tenfold decrease in the frequency of nonhomologous integration with the majority of nonhomologous integrants showing an unstable Ura⁺ phenotype. Sequencing analysis of the integration target sites showed that integration events of both ends of the integrating vector in the rad50∆ mutant occurred at different chromosomal locations, resulting in large deletions or translocations on the genomic insertion sites. Interestingly, 47% of events in the rad50∆ mutant were integrated into repetitive sequences including rDNA locus, telomeres and Ty elements and 27% of events were integrated into non-repetitive sequences as compared to 11% of events integrated into rDNA and 70% into non-repetitive sequences in the wild-type cells. These results showed that deletion of RAD50 significantly changes the distribution of different classes of integration events, suggesting that Rad50 is required for nonhomologous integration at non-repetitive sequences more so than at repetitive ones. Furthermore, Southern analysis indicated that half of the events contained deletions at one or at both ends of the integrating DNA fragment, suggesting that Rad50 might have a role in protecting free ends of double-strand breaks. In contrast to the rad50∆ mutant, the rad50S mutant (separation of function allele) slightly increases the frequency of nonhomologous integration but the distribution of integration events is similar to that of wild-type cells with the majority of events integrated into a chromosomal locus. Our results suggest that deletion of RAD50 may block the major pathway of nonhomologous integration into a non-repetitive chromosomal locus and Rad50 may be involved in tethering two ends of the integrating DNA into close proximity that facilitates nonhomologous integration of both ends into a single chromosomal locus.     

Nonhomologous end-joining deficiency allows large chromosomal deletions to be produced by replacement-type recombination in Aspergillus oryzae

Gene targeting is a technique of introducing a genetic trait at a predetermined site within a genome; it is also used to eliminate undesirable chromosomal regions from the relevant genome. Thus far, replacement-type recombination between two homologous regions separated by a large nonhomologous sequence has been hardly achieved probably due to the low frequency of homologous recombination in filamentous fungi. In this study, we report the successful and highly efficient deletion by replacement-type recombination of up to 470-kb regions of chromosome 8 and 200-kb region in chromosome 3, which includes a homologue of aflatoxin gene cluster, by nonhomologous end-joining deficient strains of Aspergillus oryzae. Our study results indicate that the deficiency of nonhomologous end-joining increases the distance of nonhomologous regions in replacement-type recombination, i.e., the possible deletion range in generation of large chromosomal deletion by one cycle of replacement-type recombination is increased in nonhomologous end-joining deficient strains.     

A test of distributive pairing in Zea mays utilizing doubly monosomic plants

Summary The v _x1 deficiency in Zea mays induces chromosomal nondisjunction during the megagametophyte divisions after meiosis producing large numbers of monosomes, trisomes, double monosomes, double trisomes, and even triple monosomes. In this study, microsporogenesis in six doubly monosomic combinations was analyzed. Double monosomes in a diploid organism provide the ideal material to determine if there is an interaction between two nonhomologous univalent chromosomes because two nonhomologous chromosomes lacking partners are present in each meiotic cell.At diakinesis and metaphase I, the two nonhomologous monosomic chromosomes were infrequently paired (3.76% and 2.18% respectively). These estimates are the upper estimates of pairing of nonhomologous monosomic chromosomes and probably represent an overestimate of these values because cells with any connections between the monosomic chromosomes were scored as having nine pairs and similar connections are not infrequently observed between two bivalents.The transmission of two nonhomologous unpaired chromosomes was deduced by studying the progeny of maize plants hyperploid for two chromosomes (a B⁴ and Wd ring). The two nonhomologous univalents disjoined randomly.Since no evidence for an interaction between nonhomologous univalent chromosomes which leads to their non-random disjunction to opposite poles was found in this study, these data confirm my earlier conclusion (Weber, 1966, 1969) that distributive pairing does not occur in maize (and probably most other plants) or that it occurs with a much lower efficiency than in Drosophila females. The frequent pairing between nonhomologous chromosomes at diakinesis and metaphase I and the non-random distribution at anaphase I in doubly trisomic maize plants reported by Michel and Burnham (1969) was found neither in my earlier studies (Weber, 1966, 1969) nor in the present study. The current study is far more sensitive than any of the previous studies because two nonhomologous chromosomes lacking pairing partners are found in every cell of a doubly monosomic plant.Research supported in part by U.S. Atomic Energy Commission Contract AT(11-1)-2121. Tech. Report No. COO-2121-10. This paper is dedicated to Professor Marcus M. Rhoades on the occasion of his 70th birthday. His guidance and help will be cherished throughout my lifetime.