Homology Recognition Research Articles

The distribution of alternating AT sequences in eukaryotic genomes suggests a role in homologous chromosome recognition in meiosis

There are general features of chromosome dynamics, such as homologue recognition in early meiosis, which are expected to involve related sequence motifs in non-coding DNA, with a similar distribution in different species. A search for such motifs is presented here. It has been carried out with the CONREPP programme. It has been found that short alternating AT sequences (10–20 bases) have a similar distribution in most eukaryotic organisms, with some exceptions related to unique meiotic features. All other microsatellite and repeat sequences vary significantly in different organisms. It is concluded that the unique structural features and uniform distribution of alternating AT sequences indicate that they may facilitate homologous chromosome pairing in the early preleptotene stage of meiosis. They may also play a role in the compaction of DNA in mitotic chromosomes.

Cohesin subunit Rad21L, the new kid on the block has new ideas

Rad21L—a new meiotic cohesin described in a recent paper in EMBO reports by the Watanabe group—probably has a role in the establishment of homologous chromosome recognition.

Dual Molecule Assay to Investigate the Mechanics of Homology Search and Recognition in Recombination

Central to homologous recombination in E. Coli is the interaction of a RecA nucleoprotein filament formed on DNA with a second DNA molecule. The filament-DNA interaction governs the search for sequence homology, sequence recognition, and eventually the exchange of homologous strands. Here, we employ a dual molecule assay to study the mechanics of this two-molecule interaction.In our experiments, a RecA filament is manipulated in 3D by dual-bead optical tweezers, and brought into contact with a second dsDNA molecule tethered in a magnetic-tweezers configuration. The strength of the interaction is studied using a force-spectroscopy technique with the magnetic bead as the sensitive force probe (resolution 10 fN/sqrt(Hz)). We present results on the interaction between supercoiled dsDNA and RecA filaments formed on dsDNA. We find that the strength of interaction depends sensitively on the supercoiling state of the dsDNA with stronger interactions recorded for negatively supercoiled DNA. This observation is interpreted as enhanced binding of DNA in a denatured state to the secondary binding site of the RecA filament. The strong affinity of the RecA filament for denatured dsDNA could lead to a favoring of the search process to regions of denatured dsDNA where base sampling is possible, a feature that is expected to be important in governing a fast and efficient homology search process.

A new meiosis-specific cohesin complex implicated in the cohesin code for homologous pairing.

We identify a new mammalian cohesin subunit, RAD21-like protein (RAD21L), with sequence similarity to RAD21 and REC8. RAD21L localizes along axial elements in early meiotic prophase, in a manner that is spatiotemporally different to either REC8 or RAD21. Remarkably, RAD21L and REC8 have symmetrical, mutually exclusive localization on the not-yet-synapsed homologues, implying that the cohesin patterning could provide a code for homologue recognition. RAD21 transiently localizes to axial elements after the dissociation of RAD21L and REC8 in late pachytene, a period of recombination repair. Further, we show that the removal of cohesins and synaptonemal complex during late meiotic prophase is promoted by Polo-like kinase 1, which is similar to the mitotic prophase pathway.

Heterology tolerance and recognition of mismatched base pairs by human Rad51 protein

Human Rad51 (hRad51) promoted homology recognition and subsequent strand exchange are the key steps in human homologous recombination mediated repair of DNA double-strand breaks. However, it is still not clear how hRad51 deals with sequence heterology between the two homologous chromosomes in eukaryotic cells, which would lead to mismatched base pairs after strand exchange. Excessive tolerance of sequence heterology may compromise the fidelity of repair of DNA double-strand breaks. In this study, fluorescence resonance energy transfer (FRET) was used to monitor the heterology tolerance of human Rad51 mediated strand exchange reactions, in real time, by introducing either G-T or I-C mismatched base pairs between the two homologous DNA strands. The strand exchange reactions were much more sensitive to G-T than to I-C base pairs. These results imply that the recognition of homology and the tolerance of heterology by hRad51 may depend on the local structural motif adopted by the base pairs participating in strand exchange. AnhRad51 mutant protein (hRad51K133R), deficient in ATP hydrolysis, showed greater heterology tolerance to both types of mismatch base pairing, suggesting that ATPase activity may be important for maintenance of high fidelity homologous recombination DNA repair.

From Strand Exchange to Branch Migration; Bypassing of Non-homologous Sequences by Human Rad51 and Rad54

Rad51 and Rad54 play crucial roles during homologous recombination. The biochemical activities of human Rad51 (hRad51) and human Rad54 (hRad54) and their interactions with each other are well documented. However, it is not known how these two proteins work together to bypass heterologous sequences; i.e. mismatched base pairs, during homologous recombination. In this study, we used a fluorescence resonance energy transfer assay to monitor homologous recombination processes in real time so that the interactions between hRad54 and hRad51 during DNA strand exchange and branch migration, which are two core steps of homologous recombination, could be characterized. Our results indicate that hRad54 can facilitate hRad51-promoted strand exchange through various degrees of mismatching. We propose that the main roles of hRad51 in homologous recombination is to initiate the homology recognition and strand-exchange steps and those of hRad54 are to promote efficient branch migration, bypass potential mismatches and facilitate long-range strand exchanges through branch migration of Holliday junctions.

Maize meiotic mutants with improper or non-homologous synapsis due to problems in pairing or synaptonemal complex formation

During meiotic prophase homologous chromosomes find each other and pair. Then they synapse, as the linear protein core (axial element or lateral element) of each homologous chromosome is joined together by a transverse central element, forming the tripartite synaptonemal complex (SC). Ten uncloned Zea mays mutants in our collection were surveyed by transmission electron microscopy by making silver-stained spreads of SCs to identify mutants with non-homologous synapsis or improper synapsis. To analyse the mutants further, zyp1, the maize orthologue of the Arabidopsis central element component ZYP1 was cloned and an antibody was made against it. Using antibodies against ZYP1 and the lateral element components AFD1 and ASY1, it was found that most mutants form normal SCs but are defective in pairing. The large number of non-homologous synapsis mutants defective in pairing illustrates that synapsis and pairing can be uncoupled. Of the ten mutants studied, only dsy2 undergoes normal homologous chromosome recognition needed for homologous pairing. The dsy2 mutation fails to maintain the SC. ZYP1 elongation is blocked at zygotene, and only dots of ZYP1 are seen at prophase I. Another mutant, mei*N2415 showed incomplete but homologous synapsis and ASY1 and AFD1 have a normal distribution. Although installation of ZYP1 is initiated at zygotene, its progression is slowed down and not completed by pachytene in some cells and ZYP1 is not retained on pachytene chromosomes. The mutants described here are now available through the Maize Genetics Cooperation Stock Center (http://maizecoop.cropsci.uiuc.edu/).

Detecting subtle functional differences in ketopantoate reductase and related enzymes using a rule-based approach with sequence-structure homology recognition scores

Ketopatoate reductase (KPR) is the second enzyme in the pantothenate (vitamin B(5)) biosynthesis pathway, an essential metabolic pathway identified as a potential target for new antimicrobials. The sequence similarity among putative KPRs is limited and KPR itself belongs to a large superfamily of 6-phosphogluconate dehydrogenases. Therefore, it is necessary to discriminate between true and other enzymes. In this paper, we describe a systematic analysis of putative KPRs in the context of this superfamily. Detailed structural analysis allowed us to define key residues for KPR activity and we classified eight structural genomics structures of the KPR family into four functional subclasses. We proposed a semi-automatic protocol, using sequence-structure homology recognition scores, for assigning KPR and related proteins to these subclasses and applied it to a representative set of 103 completely sequenced bacterial genomes. A similar approach can be applied to other enzyme families, which would aid the correct identification of drug targets and help design novel specific inhibitors.

DNA–DNA sequence homology recognition: physical mechanisms and open questions

First, we summarize recent experimental facts on homologous DNA pairing in vitro and discuss possible mechanisms of DNA-DNA sequence recognition. Then, we overview the mechanism of DNA-DNA recognition based on complementarity of DNA charge patterns. The theory predicts the recognition energy up to 10 k(B) T for close parallel homologous DNA fragments of gene-relevant lengths. We argue why this estimate cannot be directly applied to pairing of homologous DNA loci in experiments on yeast chromosomes. Namely, DNA-DNA distances assessed from experiments are much larger than those typically used in theory. Finally, we suggest some experiments that could help to judge whether short-range electrostatic forces indeed govern DNA pairing. This viewpoint paper introduces recently developed theoretical concepts to molecular biologists, with a hope to generate a junction between theory and future experiments on DNA recognition.

The molecular features of chromosome pairing at meiosis: the polyploid challenge using wheat as a reference

During meiosis, chromosome numbers are halved, leading to haploid gametes, a process that is crucial for the maintenance of a stable genome through successive generations. The process for the accurate segregation of the homologues starts in pre-meiosis as each homologue is replicated and the respective products are held together as two sister chromatids via specific cohesion proteins. At the start of meiosis, each chromosome must recognise its homologue from amongst all the chromosomes present in the nucleus and then associate or pair with that homologue. This process of homologue recognition in meiosis is more complicated in polyploids because of the greater number of related chromosomes. Despite the presence of these related chromosomes, for polyploids such as wheat to produce viable gametes, they must behave as diploids during meiosis with only true homologues pairing. In this review, the relationship between the Ph1 cyclin-dependent kinase (CDK)-like genes in wheat and the CDK2 genes in mammals and their involvement in controlling this process at meiosis is examined.

Recombination Proteins Mediate Meiotic Spatial Chromosome Organization and Pairing

Meiotic chromosome pairing involves not only recognition of homology but also juxtaposition of entire chromosomes in a topologically regular way. Analysis of filamentous fungus Sordaria macrospora reveals that recombination proteins Mer3, Msh4, and Mlh1 play direct roles in all of these aspects, in advance of their known roles in recombination. Absence of Mer3 helicase results in interwoven chromosomes, thereby revealing the existence of features that specifically ensure "entanglement avoidance." Entanglements that remain at zygotene, i.e., "interlockings," require Mlh1 for resolution, likely to eliminate constraining recombinational connections. Patterns of Mer3 and Msh4 foci along aligned chromosomes show that the double-strand breaks mediating homologous alignment have spatially separated ends, one localized to each partner axis, and that pairing involves interference among developing interhomolog interactions. We propose that Mer3, Msh4, and Mlh1 execute all of these roles during pairing by modulating the state of nascent double-strand break/partner DNA contacts within axis-associated recombination complexes.

Mutations inCaenorhabditis elegans him-19Show Meiotic Defects That Worsen with Age

From a screen for meiotic Caenorhabditis elegans mutants based on high incidence of males, we identified a novel gene, him-19, with multiple functions in prophase of meiosis I. Mutant him-19(jf6) animals show a reduction in pairing of homologous chromosomes and subsequent bivalent formation. Consistently, synaptonemal complex formation is spatially restricted and possibly involves nonhomologous chromosomes. Also, foci of the recombination protein RAD-51 occur delayed or cease altogether. Ultimately, mutation of him-19 leads to chromosome missegregation and reduced offspring viability. The observed defects suggest that HIM-19 is important for both homology recognition and formation of meiotic DNA double-strand breaks. It therefore seems to be engaged in an early meiotic event, resembling in this respect the regulator kinase CHK-2. Most astonishingly, him-19(jf6) hermaphrodites display worsening of phenotypes with increasing age, whereas defects are more severe in female than in male meiosis. This finding is consistent with depletion of a him-19-dependent factor during the production of oocytes. Further characterization of him-19 could contribute to our understanding of age-dependent meiotic defects in humans.

Physics of DNA: unravelling hidden abilities encoded in the structure of ‘the most important molecule’

A comprehensive article “Structure and Interactions of Biological Helices”, published in 2007 in Reviews of Modern Physics, overviewed various aspects of the effect of DNA structure on DNA–DNA interactions in solution and related phenomena, with a thorough analysis of the theory of these effects. Here, an updated qualitative account of this area is presented without any sophisticated ‘algebra’. It overviews the basic principles of the structure-specific interactions between double-stranded DNA and focuses on the physics behind several related properties encoded in the structure of DNA. Among them are (i) DNA condensation and aptitude to pack into small compartments of cells or viral capcids, (ii) the structure of DNA mesophases, and (iii) the ability of homologous genes to recognize each other prior to recombination from a distance. Highlighted are some of latest developments of the theory, including the shape of the ‘recognition well’. The article ends with a brief discussion of the first experimental evidence of the protein-free homology recognition in a ‘test tube’.

The most frequent short sequences in non-coding DNA

The purpose of this work is to determine the most frequent short sequences in non-coding DNA. They may play a role in maintaining the structure and function of eukaryotic chromosomes. We present a simple method for the detection and analysis of such sequences in several genomes, including Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens. We also study two chromosomes of man and mouse with a length similar to the whole genomes of the other species. We provide a list of the most common sequences of 9–14 bases in each genome. As expected, they are present in human Alu sequences. Our programs may also give a graph and a list of their position in the genome. Detection of clusters is also possible. In most cases, these sequences contain few alternating regions. Their intrinsic structure and their influence on nucleosome formation are not known. In particular, we have found new features of short sequences in C. elegans, which are distributed in heterogeneous clusters. They appear as punctuation marks in the chromosomes. Such clusters are not found in either A. thaliana or D. melanogaster. We discuss the possibility that they play a role in centromere function and homolog recognition in meiosis.

Optimizing substitution matrix choice and gap parameters for sequence alignment

BackgroundWhile substitution matrices can readily be computed from reference alignments, it is challenging to compute optimal or approximately optimal gap penalties. It is also not well understood which substitution matrices are the most effective when alignment accuracy is the goal rather than homolog recognition. Here a new parameter optimization procedure, POP, is described and applied to the problems of optimizing gap penalties and selecting substitution matrices for pair-wise global protein alignments.ResultsPOP is compared to a recent method due to Kim and Kececioglu and found to achieve from 0.2% to 1.3% higher accuracies on pair-wise benchmarks extracted from BALIBASE. The VTML matrix series is shown to be the most accurate on several global pair-wise alignment benchmarks, with VTML200 giving best or close to the best performance in all tests. BLOSUM matrices are found to be slightly inferior, even with the marginal improvements in the bug-fixed RBLOSUM series. The PAM series is significantly worse, giving accuracies typically 2% less than VTML. Integer rounding is found to cause slight degradations in accuracy. No evidence is found that selecting a matrix based on sequence divergence improves accuracy, suggesting that the use of this heuristic in CLUSTALW may be ineffective. Using VTML200 is found to improve the accuracy of CLUSTALW by 8% on BALIBASE and 5% on PREFAB.ConclusionThe hypothesis that more accurate alignments of distantly related sequences may be achieved using low-identity matrices is shown to be false for commonly used matrix types. Source code and test data is freely available from the author's web site at http://www.drive5.com/pop.

Single molecule detection of direct, homologous, DNA/DNA pairing

Using a parallel single molecule magnetic tweezers assay we demonstrate homologous pairing of two double-stranded (ds) DNA molecules in the absence of proteins, divalent metal ions, crowding agents, or free DNA ends. Pairing is accurate and rapid under physiological conditions of temperature and monovalent salt, even at DNA molecule concentrations orders of magnitude below those found in vivo, and in the presence of a large excess of nonspecific competitor DNA. Crowding agents further increase the reaction rate. Pairing is readily detected between regions of homology of 5 kb or more. Detected pairs are stable against thermal forces and shear forces up to 10 pN. These results strongly suggest that direct recognition of homology between chemically intact B-DNA molecules should be possible in vivo. The robustness of the observed signal raises the possibility that pairing might even be the "default" option, limited to desired situations by specific features. Protein-independent homologous pairing of intact dsDNA has been predicted theoretically, but further studies are needed to determine whether existing theories fit sequence length, temperature, and salt dependencies described here.

Homology recognition funnel

The recognition of homologous sequences of DNA before strand exchange is considered to be the most puzzling stage of homologous recombination. A mechanism for two homologous dsDNAs to recognize each other from a distance in electrolytic solution without unzipping had been proposed in an earlier paper [A. A. Kornyshev and S. Leikin, Phys. Rev. Lett. 86, 366 (2001)]. In that work, the difference in the electrostatic interaction energy between homologous duplexes and between nonhomologous duplexes, termed the recognition energy, has been calculated. That calculation was later extended in a series of papers to account for torsional elasticity of the molecules. A recent paper [A. A. Kornyshev and A. Wynveen, Proc. Natl. Acad. Sci. U.S.A. 106, 4683 (2009)] investigated the form of the potential well that homologous DNA molecules may feel when sliding along each other. A simple formula for the shape of the well was obtained. However, this latter study was performed under the approximation that the sliding molecules are torsionally rigid. Following on from this work, in the present article we investigate the effect of torsional flexibility of the molecules on the shape of the well. A variational approach to this problem results in a transcendental equation that is easily solved numerically. Its solutions show that at large interaxial separations the recognition well becomes wider and shallower, whereas at closer distances further unexpected features arise related to an abrupt change in the mean azimuthal alignment of the molecules. The energy surface as a function of interaxial separation and the axial shift defines what we call the recognition funnel. We show that it depends dramatically on the patterns of adsorption of counterions on DNA.

Chromosome and DNA methylation dynamics during meiosis in the autotetraploid Arabidopsis arenosa

Variation in chromosome number due to polyploidy can seriously compromise meiotic stability. In autopolyploids, the presence of more than two homologous chromosomes may result in complex pairing patterns and subsequent anomalous chromosome segregation. In this context, chromocenter, centromeric, telomeric and ribosomal DNA locus topology and DNA methylation patterns were investigated in the natural autotetraploid, Arabidopsis arenosa. The data show that homologous chromosome recognition and association initiates at telomeric domains in premeiotic interphase, followed by quadrivalent pairing of ribosomal 45S RNA gene loci (known as NORs) at leptotene. On the other hand, centromeric regions at early leptotene show pairwise associations rather than associations in fours. These pairwise associations are maintained throughout prophase I, and therefore likely to be related to the diploid-like behavior of A. arenosa chromosomes at metaphase I, where only bivalents are observed. In anthers, both cells at somatic interphase as well as at premeiotic interphase show 5-methylcytosine (5-mC) dispersed throughout the nucleus, contrasting with a preferential co-localization with chromocenters observed in vegetative nuclei. These results show for the first time that nuclear distribution patterns of 5-mC are simultaneously reshuffled in meiocytes and anther somatic cells. During prophase I, 5-mC is detected in extended chromatin fibers and chromocenters but interestingly is excluded from the NORs what correlates with the pairing pattern.

Accurate discrimination of conserved coding and non-coding regions through multiple indicators of evolutionary dynamics

BackgroundThe conservation of sequences between related genomes has long been recognised as an indication of functional significance and recognition of sequence homology is one of the principal approaches used in the annotation of newly sequenced genomes. In the context of recent findings that the number non-coding transcripts in higher organisms is likely to be much higher than previously imagined, discrimination between conserved coding and non-coding sequences is a topic of considerable interest. Additionally, it should be considered desirable to discriminate between coding and non-coding conserved sequences without recourse to the use of sequence similarity searches of protein databases as such approaches exclude the identification of novel conserved proteins without characterized homologs and may be influenced by the presence in databases of sequences which are erroneously annotated as coding.ResultsHere we present a machine learning-based approach for the discrimination of conserved coding sequences. Our method calculates various statistics related to the evolutionary dynamics of two aligned sequences. These features are considered by a Support Vector Machine which designates the alignment coding or non-coding with an associated probability score.ConclusionWe show that our approach is both sensitive and accurate with respect to comparable methods and illustrate several situations in which it may be applied, including the identification of conserved coding regions in genome sequences and the discrimination of coding from non-coding cDNA sequences.

Homology modeling and atomic level binding study of Leishmania MAPK with inhibitors

The current therapy for leishmaniasis is not sufficient and it has two severe drawbacks, host-toxicity and drug resistance. The substantial knowledge of parasite biology is not yet translating into novel drugs for leishmaniasis. Based on this observation, a 3D structural model of Leishmania mitogen-activated protein kinase (MAPK) homologue has been developed, for the first time, by homology modeling and molecular dynamics simulation techniques. The model provided clear insight in its structure features, i.e. ATP binding pocket, phosphorylation lip, and common docking site. Sequence-structure homology recognition identified Leishmania CRK3 (LCRK3) as a distant member of the MAPK superfamily. Multiple sequence alignment and 3D structure model provided the putative ATP binding pocket of Leishmania with respect to human ERK2 and LCRK3. This analysis was helpful in identifying the binding sites and molecular function of the Leishmania specific MAPK homologue. Molecular docking study was performed on this 3D structural model, using different classes of competitive ATP inhibitors of LCRK3, to check whether they exhibit affinity and could be identified as Leishmania MAPK specific inhibitors. It is well known that MAP kinases are extracellular signal regulated kinases ERK1 and ERK2, which are components of the Ras-MAPK signal transduction pathway which is complexed with HDAC4 protein, and their inhibition is of significant therapeutic interest in cancer biology. In order to understand the mechanism of action, docking of indirubin class of molecules to the active site of histone deacetylase 4 (HDAC4) protein is performed, and the binding affinity of the protein-ligand interaction was computed. The new structural insights obtained from this study are all consistent with the available experimental data, suggesting that the homology model of the Leishmania MAPK and its ligand interaction modes are reasonable. Further the comparative molecular electrostatic potential and cavity depth analysis of Leishmania MAPK and human ERK2 suggested several important differences in its ATP binding pocket. Such differences could be exploited in the future for designing Leishmania specific MAPK inhibitors.