Paul D. Sadowski

The CTCF Insulator Protein Is Posttranslationally Modified by SUMO

ABSTRACT The CTCF protein is a highly conserved zinc finger protein that is implicated in many aspects of gene regulation and nuclear organization. Its functions include the ability to act as a repressor of genes, including the c-myc oncogene. In this paper, we show that the CTCF protein can be posttranslationally modified by the small ubiquitin-like protein SUMO. CTCF is SUMOylated both in vivo and in vitro, and we identify two major sites of SUMOylation in the protein. The posttranslational modification of CTCF by the SUMO proteins does not affect its ability to bind to DNA in vitro. SUMOylation of CTCF contributes to the repressive function of CTCF on the c-myc P2 promoter. We also found that CTCF and the repressive Polycomb protein, Pc2, are colocalized to nuclear Polycomb bodies. The Pc2 protein may act as a SUMO E3 ligase for CTCF, strongly enhancing its modification by SUMO 2 and 3. These studies expand the repertoire of posttranslational modifications of CTCF and suggest roles for such modifications in its regulation of epigenetic states.

The Flp Recombinase of th 2-μm Plasmid of Saccharomyces cerevisiae

Publisher Summary Most strains of the yeast, Saccharomyces cerevisiae , harbor about 100 copies of an autonomously replicating plasmid, the 2-μm plasmid. Although the plasmid confers no advantage to its host, it is very stable. Because of its high copy-number and stability, the 2-μm plasmid has been a useful model for studying DNA replication, recombination, regulation of gene expression, and plasmid segregation in eukaryotic organisms. The plasmid has also served as the basis of many important autonomously replicating high-copy expression vectors in yeast. This chapter discusses one of the plasmids gene products, the Flp protein, which is the first eukaryotic, conservative site-specific recombinase. There are two categories of site-specific recombination: conservative site-specific recombination and transpositional recombination. Conservative site specific recombination occurs at precise sequences in DNA without any gain or loss of nucleotides. The Flp recombinase under control of a heat-shock promoter is active in Drosophila . Mosaic animals are detected after mitotic recombination among homologous chromosomes that are heterozygous for centromere-distal markers. Such mosaics are extremely useful for tracking cell fates during development. The Flp recombinase has been used in conjunction with the mobilization of P-element transposons to generate mosaics, chromosome deficiencies and duplications, and dicentric and acentric chromosomes.

Bacteriophage T7 morphogenesis: Phage-related particles in cells infected with wild-type and mutant T7 phage

Abstract Extracts from cells infected with wild-type T7 phage were found to contain three classes of particles: proheads, empty heads, and mature phage. These three particles differed with respect to their sedimentation properties, protein composition, and appearance in the electron microscope. All three structures contained the proteins specified by genes 8, 10, 13, 14, 15, and 16. Proheads and empty heads also contained the proteins coded for by genes 9 and 19. However, proheads contained more of the gene 9 product and less of the gene 19 product than empty heads. Phage did not contain the products of genes 9 or 19 but did contain three tail proteins, those specified by genes 11, 12, and 17. The product of gene 18 was found only in empty heads. In the electron microscope, proheads appeared to exclude the negative stain to a considerable degree, were round in outline, had thick shells, and contained eccentrically placed cores. Empty heads were easily penetrated by stain, had thinner shells than proheads, and often appeared to be collapsed or broken. Phage had polyhedral heads and small conical tails. Lysates of Su° cells infected with phage bearing amber mutations were examined for the presence of head-related structures. In the absence of the products of gene 9 or 10, no head-like structures were produced. After infection with a mutant in gene 8, some proheads but no empty heads or full heads were synthesized. Mutants in genes 14, 15, and 16 produced proheads, empty heads, and phage, although in reduced amounts relative to wild-type. The phage produced were noninfectious. The proheads produced after infection with mutants in genes 8, 14, 15, and 16 had aberrant protein compositions and appeared to lack cores when examined in the electron microscope. In the absence of the products of genes 18 or 19, or in the absence of T7 DNA, proheads accumulated but no other head structures were formed. After infection with mutants in genes 11, 12, and 17, head assembly and DNA packaging proceeded normally but tail assembly was defective. In the absence of the proteins specified by genes 7 and 13, phage assembly proceeded efficiently but the phage-like particles which were formed were noninfectious. On the basis of these data, we have proposed a model for phage T7 assembly in which the prohead, in the presence of the products of genes 18 and 19, packages DNA and loses P9 to give a full head to which are added the tail proteins. According to this model, the empty head is a breakdown product of a prohead which has initiated but not yet completed DNA packaging.

The Cre Recombinase Cleaves the lox Site in trans

The Cre protein is a conservative site-specific recombinase that is encoded by bacteriophage P1. Its function in vivo is to resolve dimeric lysogenic P1 plasmids that arise by general recombination. In this way Cre facilitates effective partition of the P1 prophage. Cre is a member of the integrase family of conservative site-specific recombinases. Cleavage of the DNA by the integrases involves covalent attachment of a conserved nucleophilic tyrosine to the 3′-phosphoryl end at the site of the break. We have used in vitro complementation tests to show that the Cre protein, like the Flp protein of the 2-μm plasmid of Saccharomyces cerevisiae, cleaves its target lox site in trans. Moreover, the data are compatible with two modes of cleavage; one requires the reconstitution of a pseudo full-site from half-sites and the other requires the assembly of a higher order complex that resembles a synaptic complex.

Constitutional UPD for chromosome 11p15 in individuals with isolated hemihyperplasia is associated with high tumor risk and occurs following assisted reproductive technologies

Isolated hemihyperplasia (IH) refers to a distinct diagnosis involving asymmetric overgrowth of single or multiple organs or regions of the body and can result from various genomic changes including molecular alterations of 11p15; these are paternal uniparental disomy (UPD), and alterations of methylation at two imprinting centers at 11p15: IC1 (H19) and IC2 (KCNQ1OT1). As little information is available on the molecular basis of tumor development in IH, or on the frequency of tumors in children with different molecular subtypes of IH, molecular testing was undertaken on 51 patients with IH and revealed: 8 (16%) with UPD, 3 (6%) with hypomethylation at KCNQ1OT1, and 0 with hypermethylation at H19. Of the 8 patients with UPD, 4 had tumors (3 hepatoblastomas, 1 Wilms tumor); 0/3 patients with hypomethylation at KCNQ1OT1 had a tumor; of the remaining 40 with no molecular alterations, 6 had tumors (3 Wilms tumors, 2 neuroblastomas, 1 adrenocortical adenoma). The 50% tumor frequency in patients with IH and UPD was statistically significantly higher than the 15% tumor frequency in those with IH and no molecular alteration detected (Fishers exact test P = 0.047, OR 5.67). This is the first demonstration that UPD at 11p15 in patients with IH confers a higher tumor risk than in patients with IH without this molecular change. Of note, two of the eight patients with UPD and IH were conceived using assisted reproductive technologies (ART), thus raising the question whether ART might impact the rate of somatic recombination during embryonic development.

The involvement of genes 3,4,5 and 6 in genetic recombination in bacteriophage T7.

Abstract The effect of conditionally lethal mutations in various genes of phage T7 on genetic recombination was determined by doing genetic crosses under nonpermissive conditions. The experiments have implicated the products of gene 3 (T7 endonuclease), gene 4 (DNA replication protein), gene 5 (T7 DNA polymerase) and gene 6 (T7 exonuclease) in phage T7 genetic recombination.

The Crystal Structure of Bacteriophage HK97 gp6: Defining a Large Family of Head-Tail Connector Proteins

The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 A crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophage SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.

Renal Abnormalities in Beckwith-Wiedemann Syndrome Are Associated with 11p15.5 Uniparental Disomy

Beckwith-Wiedemann syndrome (BWS) is a somatic overgrowth syndrome characterized by a variable incidence of congenital anomalies, including hemihyperplasia and renal malformations. BWS is associated with disruption of genomic imprinting and/or mutations in one or more genes encoded on 11p15.5, including CDKN1C (p57(KIP2)). It was hypothesized that genotypic and epigenotypic abnormalities of the 11p15.5 region affecting CDKN1C were associated with renal abnormalities. Medical records for 159 individuals with BWS were reviewed. All underwent at least one abdominal ultrasonographic evaluation. Testing for paternal uniparental disomy (UPD) at 11p15.5, CDKN1C mutations, and imprinting defects at KvDMR1 was performed for 96, 32, and 47 patients, respectively. Of the 159 patients, 67 (42%) exhibited renal abnormalities, mainly nephromegaly (25%), collecting system abnormalities (11%), and renal cysts (10.5%). The frequency of renal lesions among patients who were tested for genetic abnormalities did not differ from that among patients who were not tested. Paternal UPD was demonstrated in 22 of 96 cases (23%), CDKN1C mutations in eight of 32 cases (25%), and KvDMR1 imprinting defects in 21 of 47 cases (45%). The 22 UPD-positive patients exhibited a significantly higher incidence of renal abnormalities (P = 0.0026). Surprisingly, the eight patients with CDKN1C mutations exhibited no significant increase in the incidence of renal lesions (P = 0.29). Imprinting defects at KvDMR1, which might downregulate CDKN1C, were also not associated with a significant difference in the incidence of renal disease. Whereas UPD at 11p15.5 in BWS was associated with a higher incidence of renal abnormalities, mutations at CDKN1C and KvDMR1 imprinting defects were not, suggesting that imprinted genes on 11p15.5 other than CDKN1C are critical for renal development.

Isolation of intermediates in the binding of the FLP recombinase of the yeast plasmid 2-micron circle to its target sequence☆

We describe a method for isolating and characterizing intermediates in the binding of the FLP recombinase, encoded by the yeast plasmid 2-micron circle to its target sequence. On a wild-type substrate, three specific complexes are formed. Footprinting analysis of the gel-purified complexes shows that each complex is the result of a unique FLP-DNA association. On the basis of the behavior of various FLP target sequences in the gel-binding assay, we propose a model describing the steps that lead to the formation of a stable FLP-DNA complex.

Mutagenesis of a conserved region of the gene encoding the FLP recombinase of Saccharomyces cerevisiae: A role for arginine 191 in binding and ligation☆

The FLP recombinase from the 2 microns plasmid of Saccharomyces cerevisiae contains a region from amino acid 185 to 203 that is conserved among several FLP-like proteins from different yeasts. Using site-directed mutagenesis, we have made mutations in this region of the FLP gene. Five of twelve mutations in the region yielded proteins that were unable to bind to the FLP recombination target (FRT) site. A change of arginine at position 191 to lysine resulted in a protein (FLP-R191K) that could bind to the FRT site but could not catalyze recombination. This mutant protein accumulated as a stable protein-DNA complex in which one of the two bound FLP proteins was covalently attached to the DNA. FLP-R191K was defective in strand exchange and ligation and was unable to promote protein-protein interaction with half-FRT sites. The conservation of three residues in all members of the integrase family of site-specific recombinases (His305, Arg308, Tyr343 in FLP) implies a common mechanism of recombination. The conservation of arginine 191 and the properties of the FLP-R191K mutant protein suggest that this arginine also plays an important role in the mechanism of FLP-mediated site-specific recombination.