The complex binding of PRDM9

A recent study investigates the in vitro DNA binding behavior of PRDM9, a zinc finger protein involved in the localization of recombination hotspots in mammals.Please see related research article: http://genomebiology.com/2013/14/4/R35     

PRDM9在哺乳动物基因重组热点中的作用

PRDM9(PR domain containing 9)是一种可催化组蛋白H3的4位赖氨酸(H3K4)发生三甲基化修饰的甲基转移酶,还拥有转录因子活性．PRDM9主要在生殖细胞的减数分裂初期表达,它的功能异常可导致不育的发生．哺乳动物PRDM9在C端的锌指结构具有进化快速的特征,而这个特征与基因重组热点的多样性相对应,一系列证据表明,PRDM9参与了基因重组热点的结合和重组的起始．这些进展对深入理解物种进化和基因重组机制具有十分重要的意义．     

PRDM9 Methyltransferase Activity Is Essential for Meiotic DNA Double-Strand Break Formation at Its Binding Sites

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Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY

Bacteriophage T4 UvsY is a recombination mediator protein that promotes assembly of the UvsX-ssDNA presynaptic filament. UvsY helps UvsX to displace T4 gene 32 protein (gp32) from ssDNA, a reaction necessary for proper formation of the presynaptic filament. Here we use DNA stretching to examine UvsY interactions with single DNA molecules in the presence and absence of gp32 and a gp32 C-terminal truncation (*I), and show that in both cases UvsY is able to destabilize gp32-ssDNA interactions. In these experiments UvsY binds more strongly to dsDNA than ssDNA due to its inability to wrap ssDNA at high forces. To support this hypothesis, we show that ssDNA created by exposure of stretched DNA to glyoxal is strongly wrapped by UvsY, but wrapping occurs only at low forces. Our results demonstrate that UvsY interacts strongly with stretched DNA in the absence of other proteins. In the presence of gp32 and *I, UvsY is capable of strongly destabilizing gp32-DNA complexes in order to facilitate ssDNA wrapping, which in turn prepares the ssDNA for presynaptic filament assembly in the presence of UvsX. Thus, UvsY mediates UvsX binding to ssDNA by converting rigid gp32-DNA filaments into a structure that can be strongly bound by UvsX.     

A RAD52‐like single‐stranded DNA binding protein affects mitochondrial DNA repair by recombination

The plant mitochondrial DNA‐binding protein ODB1 was identified from a mitochondrial extract after DNA‐affinity purification. ODB1 (organellar DNA‐binding protein 1) co‐purified with WHY2, a mitochondrial member of the WHIRLY family of plant‐specific proteins involved in the repair of organellar DNA. The Arabidopsis thaliana ODB1 gene is identical to RAD52‐1, which encodes a protein functioning in homologous recombination in the nucleus but additionally localizing to mitochondria. We confirmed the mitochondrial localization of ODB1 by in vitro and in vivo import assays, as well as by immunodetection on Arabidopsis subcellular fractions. In mitochondria, WHY2 and ODB1 were found in large nucleo‐protein complexes. Both proteins co‐immunoprecipitated in a DNA‐dependent manner. In vitro assays confirmed DNA binding by ODB1 and showed that the protein has higher affinity for single‐stranded than for double‐stranded DNA. ODB1 showed no sequence specificity in vitro. In vivo, DNA co‐immunoprecipitation indicated that ODB1 binds sequences throughout the mitochondrial genome. ODB1 promoted annealing of complementary DNA sequences, suggesting a RAD52‐like function as a recombination mediator. Arabidopsis odb1 mutants were morphologically indistinguishable from the wild‐type, but following DNA damage by genotoxic stress, they showed reduced mitochondrial homologous recombination activity. Under the same conditions, the odb1 mutants showed an increase in illegitimate repair bypasses generated by microhomology‐mediated recombination. These observations identify ODB1 as a further component of homologous recombination‐dependent DNA repair in plant mitochondria.     

Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer

Zinc-finger–FokI nucleases (ZFNs) are useful for manipulating genomic DNA, but two ZFNs are required to cleave one site of double-stranded DNA (dsDNA), which limits the choice of targets. To refine ZFN technology, we constructed artificial zinc-finger nucleases containing an artificial zinc-finger protein (AZP) and a single-chain FokI dimer with nine different peptide linkers between two FokI molecules (designated AZP–scFokI). DNA cleavage assays revealed that the AZP–scFokI variant possessing the longest peptide linker cleaved dsDNA with equal or greater reactivity than the corresponding AZP–FokI dimer. The DNA cleavage pattern of AZP–scFokI suggests that the enhanced dsDNA cleavage was due to increased formation of FokI dimer in AZP–scFokI. Furthermore, we demonstrated that AZP–scFokI site-specifically cleaved its target DNA due to the AZP moiety discriminating one base pair difference. Thus, a single AZP–scFokI molecule is able to cleave dsDNA efficiently and site-specifically, and enhances the usefulness of the ZFN approach.     

Prdm9 and Meiotic Cohesin Proteins Cooperatively Promote DNA Double-Strand Break Formation in Mammalian Spermatocytes

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A nuclear matrix protein related to intermediate filaments proteins is a member of the complex binding alphoid DNA in vitro

A complex of three proteins (of 80, 70, 58 kDa-p80, p70, and p58, respectively) with the ability to bind alphoid DNA (alpha-satDNA) was revealed by gel mobility shift assay (GMSA) in human nuclear matrix. The probes of the alpha-satDNA bound in the GMSA with the greatest specificity, but the complex was capable of binding human satellite 3 fragment. According to ion exchange and affinity chromatography, the complex includes two DNA-binding proteins, p70 and p80, and a non-DNA-binding one, p58, which enhances the specificity of binding to the alpha-satDNA. GMSA, SDS-PAGE and immunoblotting showed that the lamins, as well as constitutive centromeric proteins (CENP-A, CENP-B, CENP-C, CENP-G), were not incorporated into the complex. It was demonstrated by immunoprecipitation assay that p70 and, probably p58, share a common antigen determinant with the rod domain of intermediate filaments (IF) proteins. The results obtained indicate that the nuclear matrix contains at least one IF-related protein that is able to bind specifically to alpha-satDNA in vitro and that this protein is distinct from the lamins.     

DNA binding of a non-sequence-specific HMG-D protein is entropy driven with a substantial non-electrostatic contribution

The thermal properties of two forms of the Drosophila melanogaster HMG-D protein, with and without its highly basic 26 residue C-terminal tail (D100 and D74) and the thermodynamics of their non-sequence-specific interaction with linear DNA duplexes were studied using scanning and titration microcalorimetry, spectropolarimetry, fluorescence anisotropy and FRET techniques at different temperatures and salt concentrations. It was shown that the C-terminal tail of D100 is unfolded at all temperatures, whilst the state of the globular part depends on temperature in a rather complex way, being completely folded only at temperatures close to 0 degrees C and unfolding with significant heat absorption at temperatures below those of the gross denaturational changes. The association constant and thus Gibbs energy of binding for D100 is much greater than for D74 but the enthalpies of their association are similar and are large and positive, i.e. DNA binding is a completely entropy-driven process. The positive entropy of association is due to release of counterions and dehydration upon forming the protein/DNA complex. Ionic strength variation showed that electrostatic interactions play an important but not exclusive role in the DNA binding of the globular part of this non-sequence-specific protein, whilst binding of the positively charged C-terminal tail of D100 is almost completely electrostatic in origin. This interaction with the negative charges of the DNA phosphate groups significantly enhances the DNA bending. An important feature of the non-sequence-specific association of these HMG boxes with DNA is that the binding enthalpy is significantly more positive than for the sequence-specific association of the HMG box from Sox-5, despite the fact that these proteins bend the DNA duplex to a similar extent. This difference shows that the enthalpy of dehydration of apolar groups at the HMG-D/DNA interface is not fully compensated by the energy of van der Waals interactions between these groups, i.e. the packing density at the interface must be lower than for the sequence-specific Sox-5 HMG box.     

Prediction and validation of the unexplored RNA‐binding protein atlas of the human proteome

Detecting protein‐RNA interactions is challenging both experimentally and computationally because RNAs are large in number, diverse in cellular location and function, and flexible in structure. As a result, many RNA‐binding proteins (RBPs) remain to be identified. Here, a template‐based, function‐prediction technique SPOT‐Seq for RBPs is applied to human proteome and its result is validated by a recent proteomic experimental discovery of 860 mRNA‐binding proteins (mRBPs). The coverage (or sensitivity) is 42.6% for 1217 known RBPs annotated in the Gene Ontology and 43.6% for 860 newly discovered human mRBPs. Consistent sensitivity indicates the robust performance of SPOT‐Seq for predicting RBPs. More importantly, SPOT‐Seq detects 2418 novel RBPs in human proteome, 291 of which were validated by the newly discovered mRBP set. Among 291 validated novel RBPs, 61 are not homologous to any known RBPs. Successful validation of predicted novel RBPs permits us to further analysis of their phenotypic roles in disease pathways. The dataset of 2418 predicted novel RBPs along with confidence levels and complex structures is available at http://sparks-lab.org (in publications) for experimental confirmations and hypothesis generation. Proteins 2014; 82:640–647. © 2013 Wiley Periodicals, Inc.     

Identification of residues important for DNA binding in the full-length human Rad52 protein

Human Rad52 (HsRad52) is a DNA-binding protein (418 residues) that promotes the catalysis of DNA double strand break repair by the Rad51 recombinase. HsRad52 self-associates to form ring-shaped oligomers as well as higher order complexes of these rings. Analysis of the structural and functional organization of protein domains suggests that many of the determinants of DNA binding lie within the N-terminal 85 residues. Crystal structures of two truncation mutants, HsRad52(1-212) and HsRad52(1-209) support the idea that this region makes up an important part of the DNA binding domain. Here, we report the results of saturating alanine scanning mutagenesis of the N-terminal domain of full-length HsRad52 in which we identify residues that are likely involved in direct contact with single-stranded DNA (ssDNA). Our results largely agree with the position of side-chains seen in the crystal structures but also suggest that certain DNA binding and cross-subunit interactions differ between the 11 subunit ring in the crystal structures of the truncation mutant proteins versus the seven subunit ring formed by full-length HsRad52.     

Evidence of positive selection and concerted evolution in the rapidly evolving PRDM9 zinc finger domain in goats and sheep

Meiotic recombination contributes to augmentation of genetic diversity, exclusion of deleterious alleles and proper segregation of chromatids. PRDM9 has been identified as the gene responsible for specifying the location of recombination hotspots during meiosis and is also the only known vertebrate gene associated with reproductive isolation between species. PRDM9 encodes a protein with a highly variable zinc finger (ZF) domain that varies between as well as within species. In the present study, the ZF domain of PRDM9 on chromosome 1 was characterized for the first time in 15 goat breeds and 25 sheep breeds of India. A remarkable variation in the number and sequence of ZF domains was observed. The number of ZF repeats in the ZF array varied from eight to 12 yielding five homozygous and 10 heterozygous genotypes. The number of different ZF domains was 84 and 52 producing 36 and 26 unique alleles in goats and sheep respectively. The posterior mean of dN/dS or omega values were calculated using the codeml tool of pamlx to identify amino acids that are evolving positively in goats and sheep, as positions ?1, +3 and +6 in the ZF domain have been reported to experience strong positive selection across different lineages. Our study identified sites ?5, ?1, +3, +4 and +6 to be experiencing positive selection. Small ruminant zinc fingers were also found to be evolving under concerted evolution. Our results demonstrate the existence of a vast diversity of PRDM9 in goats and sheep, which is in concert with reports in many metazoans.     

Molecular beacons for detecting DNA binding proteins: mechanism of action

New methodology for detecting sequence-specific DNA binding proteins has been recently developed (T. Heyduk, and E. Heyduk, Nat. Biotechnol. 20 (2002) 171). The central feature of this assay is protein-dependent association of two DNA fragments, each containing about half of a DNA sequence-defining the protein binding site. In this report we propose a physical model explaining the functioning of the assay. The model involves two linked equilibria: association between the two DNA fragments and binding of the protein exclusively to the complex between the two DNA fragments. Equilibrium and kinetic experiments provided evidence supporting the proposed model and showed that the model was sufficient to describe the behavior of the assay under a variety of conditions. Kinetic data identified the association between the two DNA half-sites as the rate-limiting step of the assay. Theoretical simulations based on the proposed model were used to investigate parameters important for the maximal sensitivity of the assay. Physical understanding of the assay will provide means for rational design of the assay for a variety of target proteins.     

Induced DNA recombination by Cre recombinase protein transduction

Cre is a DNA recombinase that recognizes 34 base-pair loxP sites of recombination. We have developed a cell-permeable Cre recombinase, TATCre, that is capable of mediating deletion of loxP-flanked targets by simply adding TATCre to cell cultures. Thus, TATCre allows efficient induced DNA recombination without the use of a Cre recombinase transgene or any other genetic material and should prove useful for the genetic manipulation of a wide variety of cell types that have been engineered to possess loxP sites.     

DNA deformation energetics and protein binding

The formation of protein-DNA complexes often involves deformation of the DNA double helix. We have calculated the energy necessary to produce this deformation in 71 crystallographically determined complexes, using internal coordinate energy optimization with the JUMNA program and a generalized Born continuum solvent treatment. An analysis of the data allows deformation energy to be interpreted in terms of both local and global structural changes. We find that, in the majority of complexes, roughly 60% of the deformation energy corresponds to backbone distortion. It is also found that large changes in stacking and pairing energies are often compensated for by other, longer range, stabilizing factors. Some deformations, such as base opening, can be large, but only-produce local energetic effects. In terms of backbone distortions, the angle alpha, most often involved in alphagamma transitions, makes the most significant energetic contribution. This type of transition is twice as costly as those involving beta, or coupled epsilonzeta changes. Sugar amplitude changes are also energetically significant, in contrast to changes in phase angles.     

Wing 1 of protein HOP2 is as important as helix 3 in DNA binding by MD simulation

The repair of programmed DNA double-strand breaks through recombination is required for proper association and disjunction of the meiotic homologous chromosomes. Meiosis-specific protein HOP2 plays essential roles in recombination by promoting recombinase activities. The N-terminal domain of HOP2 interacts with DNA through helix 3 (H3) and wing 1 (W1). Mutations in wing 1 (Y65A/K67A/Q68A) slightly weakened the binding but mutations in helices 2 and 3 (Q30A/K44A/K49A) nearly abolished the binding. To better understand such differential effects at atomic level, molecular dynamics simulations were employed. Despite losing some hydrogen bonds, the W1-mutant DNA complex was rescued by stronger hydrophobic interactions. For the wild type and W1-mutant, the protein was found to slide along the DNA grooves as the DNA rolls along its double-helix axis. This motion could be functionally important to facilitate the precise positioning of the single-stranded DNA with the homologous double-stranded DNA. The sliding motion was reduced in the W1-mutant. The H-mutant nearly lost all intermolecular interactions. Moreover, an additional mutation in wing 1 (Y65A/K67A/Q68A/K69A) also caused complete complex dissociation. Therefore, both wing 1 and helix 3 make important contribution to the DNA binding, which could be important to the strand invasion function of HOP2 homodimer and HOP2-MND1 heterodimer. Similar to cocking a medieval crossbow with the archer’s foot placed in the stirrup, wing 1 may push the minor groove to cause distortion while helix 3 grabs the major groove.     

The role and mechanism of long non-coding RNAs in homologous recombination repair of radiation-induced DNA damage

DNA double-strand breaks can seriously damage the genetic information that organisms depend on for survival and reproduction. Therefore, cells require a robust DNA damage response mechanism to repair the damaged DNA. Homologous recombination (HR) allows error-free repair, which is key to maintaining genomic integrity. Long non-coding RNAs (lncRNAs) are RNA molecules that are longer than 200 nucleotides. In recent years, a number of studies have found that lncRNAs can act as regulators of gene expression and DNA damage response mechanisms, including HR repair. Moreover, they have significant effects on the occurrence, development, invasion and metastasis of tumor cells, as well as the sensitivity of tumors to radiotherapy and chemotherapy. These studies have therefore begun to expose the great potential of lncRNAs for clinical applications. In this review, we focus on the regulatory roles of lncRNAs in HR repair.