DNA binding specificities of the long zinc-finger recombination protein PRDM9

Background

Meiotic recombination ensures proper segregation of homologous chromosomes and creates genetic variation. In many organisms, recombination occurs at limited sites, termed ''hotspots'', whose positions in mammals are determined by PR domain member 9 (PRDM9), a long-array zinc-finger and chromatin-modifier protein. Determining the rules governing the DNA binding of PRDM9 is a major issue in understanding how it functions.

Results

Mouse PRDM9 protein variants bind to hotspot DNA sequences in a manner that is specific for both PRDM9 and DNA haplotypes, and that in vitro binding parallels its in vivo biological activity. Examining four hotspots, three activated by Prdm9^Cst and one activated by Prdm9^Dom2, we found that all binding sites required the full array of 11 or 12 contiguous fingers, depending on the allele, and that there was little sequence similarity between the binding sites of the three Prdm9^Cst activated hotspots. The binding specificity of each position in the Hlx1 binding site, activated by Prdm9^Cst, was tested by mutating each nucleotide to its three alternatives. The 31 positions along the binding site varied considerably in the ability of alternative bases to support binding, which also implicates a role for additional binding to the DNA phosphate backbone.

Conclusions

These results, which provide the first detailed mapping of PRDM9 binding to DNA and, to our knowledge, the most detailed analysis yet of DNA binding by a long zinc-finger array, make clear that the binding specificities of PRDM9, and possibly other long-array zinc-finger proteins, are unusually complex.     

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

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Structural study of the N-terminal domain of human MCM8/9 complex

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

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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.     

锌指蛋白核酸酶的作用原理及其应用

锌指蛋白核酸酶(Zinc finger nucleases,ZFN)因其能特异性识别并切割DNA序列以及可设计性,被用于基因定点突变和外源基因定点整合。目前,ZFN技术以其准确的靶位点设计能力和诱发高效率基因打靶的优势,越来越受到基因改造研究者的重视,已经成功应用于动植物细胞、胚胎的基因改造。随着鉴定靶DNA高亲和力的锌指蛋白(Zinc finger protein,ZFP)实验技术日渐成熟,可以预见到不久的将来这项技术会在基因工程和育种中得到广泛应用。文章介绍了锌指蛋白识别DNA靶位点和ZFN介导的基因打靶(Double strand break gene targeting,DSB-GT)的原理,同时还综述了目前ZFN技术用于基因改造的研究进展。     

Molecular strategies to replace the structural metal site in the prokaryotic zinc finger domain

The specific arrangement of secondary elements in a local motif often totally relies on the formation of coordination bonds between metal ions and protein ligands. This is typified by the ~ 30 amino acid eukaryotic zinc finger motif in which a β-sheet and an α-helix are clustered around a zinc ion by various combinations of four ligands.     

Positive and negative cooperativity of modularly assembled zinc fingers

One simple and widespread method to create engineered zinc fingers targeting the desired DNA sequences is to modularly assemble multiple finger modules pre-selected to recognize each DNA triplet. However, it has become known that a sufficient DNA binding affinity is not always obtained. In order to create successful zinc finger proteins, it is important to understand the context-dependent contribution of each finger module to the DNA binding ability of the assembled zinc finger proteins. Here, we have created finger-deletion mutants of zinc finger proteins and examined the DNA bindings of these zinc fingers to clarify the contributions of each finger module. Our results indicate that not only a positive cooperativity but also a context-dependent reduction in the DNA binding activity can be induced by assembling zinc finger modules.     

PRDM在生殖细胞发育中的作用

生殖细胞特化是发育和遗传的基础。原始生殖细胞（精子和卵子的前体细胞）的特化包括3个主要事件：体细胞程序的抑制、潜在全能性的获得、基因组范围内的表观遗传重编程。含PR域蛋白1（PR domain-containing1,PRDM1）和PRDM14是生殖细胞系产生的关键转录调节因子。PRDMl要抑制体细胞程序,而PRDM14主要调节潜在全能性的获得及表观遗传学重编程。此外,PRDM家族蛋白PRDM9在生殖细胞减数分裂中有重要作用。     

The MCM8/9 complex: A recent recruit to the roster of helicases involved in genome maintenance

There are several DNA helicases involved in seemingly overlapping aspects of homologous and homoeologous recombination. Mutations of many of these helicases are directly implicated in genetic diseases including cancer, rapid aging, and infertility. MCM8/9 are recent additions to the catalog of helicases involved in recombination, and so far, the evidence is sparse, making assignment of function difficult. Mutations in MCM8/9 correlate principally with primary ovarian failure/insufficiency (POF/POI) and infertility indicating a meiotic defect. However, they also act when replication forks collapse/break shuttling products into mitotic recombination and several mutations are found in various somatic cancers. This review puts MCM8/9 in context with other replication and recombination helicases to narrow down its genomic maintenance role. We discuss the known structure/function relationship, the mutational spectrum, and dissect the available cellular and organismal data to better define its role in recombination.     

DNA inhibits catalysis by the carboxyltransferase subunit of acetyl-CoA carboxylase: implications for active site communication

Acetyl-CoA carboxylase (ACC) catalyzes the first committed step in the synthesis of long-chain fatty acids. The crystal structure of the Escherichia coli carboxyltransferase component of ACC revealed an alpha(2)beta(2) subunit composition with two active sites and, most importantly, a unique zinc domain in each alphabeta pair that is absent in the eukaryotic enzyme. We show here that carboxyltransferase binds DNA. Half-maximal saturation of different single-stranded or double-stranded DNA constructs is seen at 0.5-1.0 muM, and binding is cooperative and nonspecific. The substrates (malonyl-CoA and biocytin) inhibit DNA:carboxyltransferase complex formation. More significantly, single-stranded DNA, double-stranded DNA, and heparin inhibit the reaction catalyzed by carboxyltransferase, with single-stranded DNA and heparin acting as competitive inhibitors. However, double-inhibition experiments revealed that both DNA and heparin can bind the enzyme in the presence of a bisubstrate analog (BiSA), and the binding of BiSA has a very weak synergistic effect on the binding of the second inhibitor (DNA or heparin) and vice versa. In contrast, DNA and heparin can also bind to the enzyme simultaneously, but the binding of either molecule has a strong synergistic effect on binding of the other. An important mechanistic implication of these observations is that the dual active sites of ACC are functionally connected.     

siRNA screening identifies METTL9 as a histidine Nπ-methyltransferase that targets the proinflammatory protein S100A9

Protein methylation is one of the most common post-translational modifications observed in basic amino acid residues, including lysine, arginine, and histidine. Histidine methylation occurs on the distal or proximal nitrogen atom of its imidazole ring, producing two isomers: Nτ-methylhistidine or Nπ-methylhistidine. However, the biological significance of protein histidine methylation remains largely unclear owing in part to the very limited knowledge about its contributing enzymes. Here, we identified mammalian seven-β-strand methyltransferase METTL9 as a histidine Nπ-methyltransferase by siRNA screening coupled with methylhistidine analysis using LC–tandem MS. We demonstrated that METTL9 catalyzes Nπ-methylhistidine formation in the proinflammatory protein S100A9, but not that of myosin light chain kinase MYLK2, in vivo and in vitro. METTL9 does not affect the heterodimer formation of S100A9 and S100A8, although Nπ-methylation of S100A9 at His-107 overlaps with a zinc-binding site, attenuating its affinity for zinc. Given that S100A9 exerts an antimicrobial activity, probably by chelation of zinc essential for the growth of bacteria and fungi, METTL9-mediated S100A9 methylation might be involved in the innate immune response to bacterial and fungal infection. Thus, our findings suggest a functional consequence for protein histidine Nπ-methylation and may add a new layer of complexity to the regulatory mechanisms of post-translational methylation.