Minisatellite binding protein Msbp-1 is a sequence-specific single-stranded DNA-binding protein.

Msbp-1 is a minisatellite-specific DNA-binding protein. Using synthetic binding substrates, we now show that Msbp-1 binds not to double-stranded DNA, but exclusively to single-stranded DNA. Binding is specific to the guanine-rich strand of the minisatellite duplex, interactions with the cytosine-rich strand being undetectable by southwestern analysis. Furthermore, the binding site required for successful DNA-protein interactions appears to be two or more minisatellite repeat units. We have also isolated, by whole-genome PCR and cloning, one Msbp-1 binding site from the human genome. Again, the binding strand of this molecule contains a repetitive G-rich structure equivalent to that of a small minisatellite. These observations are discussed with respect to other single-stranded DNA-binding proteins known to play a role in recombination processes.     

LRP130, a protein containing nine pentatricopeptide repeat motifs, interacts with a single-stranded cytosine-rich sequence of mouse hypervariable minisatellite Pc-1.

Recently, we have identified and purified minisatellite DNA binding proteins (MNBPs) that bind to the mouse hypervariable minisatellite Pc-1, from NIH3T3 cells. This study describes the isolation and characterization of a mouse leucine-rich protein (mLRP130) as one of the MNBPs that binds to the C-rich strand of Pc-1. The mLRP130 cDNA was demonstrated to encode a polypeptide of 1306 amino-acid residues with a deduced molecular mass of 137 kDa, and the mLRP130 mRNA is detected in various organs, including heart, brain, liver, skeletal muscle, kidneys and testes. The mLRP130 protein has nine copies of pentatricopeptide repeat (PPR) motifs that are considered to serve as protein-protein interactions. Two forms of the mLRP130 protein were detected in NIH3T3 cells with an approximate molecular mass of 140 kDa (mLRP130) and 100 kDa (mLRP130der), and were detected mainly in nuclear and cytoplasmic fractions, respectively. Immunofluorescence microscopic analysis demonstrated dominant localization of mLRP130 at the perinuclear region, and also in the nucleus and cytoplasm with dot- or squiggle-like staining. The immunoprecipitated mLRP130 bound to the single-stranded d(CTGCC)8, but not to its complementary G-rich strand of d(GGCAG)8 or double-stranded form. Possible biological roles of mLRP130 are discussed in association with the stability of minisatellite DNA sequences.     

Two hypervariable minisatellite DNA binding proteins.

Hypervariable minisatellite DNA sequences are short, tandemly repeated sequences present at numerous loci in eukaryotes. They stimulate intermolecular homologous recombination up to 13-fold in human cells in culture and may be specific sites for the initiation of recombination in the eukaryotic genome (Wahls, W.P., Wallace, L.J., & Moore, P.D. (1990) Cell 60, 95-103). Reported here is the detection and partial purification of two hypervariable minisatellite DNA binding proteins, called Msbp-2 and Msbp-3, present in the nuclear extracts of human HeLa cells. The proteins elute from a gel filtration column with a native mass of 200-250 kDa and have sizes of 77 kDa and 115 kDa respectively.     

Detection and isolation of minisatellite Pc-1 binding proteins.

Minisatellites (MNs) are arrays of 5-100 nucleotide repeats that are dispersed throughout the genome of vertebrates. They demonstrate alteration in tumors and in cells exposed to various carcinogens, but the molecular mechanisms underlying the induction of mutations at MNs are largely unknown. Hypervariable MN Pc-1 isolated from the mouse genome consists of tandem repeats of d(GGCAG) flanked with locus-specific sequences at both ends. We have found that MN mutations are induced in NIH3T3 cells by treatment with okadaic acid using a Pc-1 MN fragment as a probe. In order to shed light on the molecular mechanisms, we isolated six MN Pc-1 binding proteins, pA, pB, pD, pE, pF and pG, from nuclear extracts of NIH3T3 cells treated with okadaic acid. While pA and pB bound to the G-rich strand of Pc-1, pD, pE, pF and pG bound to the complementary C-rich strand. Sequence specificities for DNA binding were revealed and one base substitution and insertion into the Pc-1 repeat unit dramatically changed the affinity of each protein, suggesting that they bind to Pc-1 and Pc-1-like MNs in vivo.     

Detection of a novel minisatellite-specific DNA-binding protein.

We describe the detection of a ubiquitous DNA-binding protein which appears to interact specifically with tandem-repeated minisatellites. The murine 40 kd protein, which we term Msbp-1, was found to be present in all mouse tissues tested. This protein was bound specifically and with high affinity by double-stranded DNA containing a repeat sequence related to the minisatellite 'core' sequence, and binding required the presence of multiple repeat units. Corresponding minisatellite-specific DNA-binding proteins could also be detected in species ranging from Drosophila to man. This analysis represents the first direct evidence that minisatellites can function as a specific recognition signal for an endogenous DNA-binding protein.     

Effect of SCID mutation on the occurrence of mouse Pc-1 (Ms6-hm) germline mutations

Mouse Pc-1 (Ms6-hm) is a hypervariable minisatellite locus that is unstable during intergenerational transmission. This hyper-instability of Pc-1 is useful for detecting germline mutation using a small number of experimental animals, although its molecular mechanism has not yet been elucidated. We examined the effect of severe combined immune deficiency (SCID) mutation on the spontaneous germline mutation at the Pc-1 locus using the CB17 mouse strain. Our results showed that the frequency of spontaneous germline mutation at Pc-1 in the offspring of wild-type parents was 9.7%. In F1 between SCID male and wild-type female, however, the frequency of germline mutation was drastically increased to 42.3%. When SCID female mice were mated with wild-type male, the frequency of germline mutation in F1 was slightly increased to 13.6%. These results suggest that DNA protein kinase catalytic subunit (DNA-PKcs), deficiency of which causes SCID mutation, plays an important role in the stable transmission of a genome containing hypervariable tandem repeats to progeny in male germ cells.     

Effects of lichen heteroglycans on proliferation and IL-10 secretion by rat spleen cells and IL-10 and TNF-alpha secretion by rat peritoneal macrophages in vitro.

Four polysaccharides Pc-1, Pc-2, Pc-3 and Pc-4 were isolated from water and alkali extracts of the lichen Peltigera canina using ethanol fractionation, gel filtration and preparative HP-GPC. The monosaccharide composition was determined by methanolysis and GC and showed mannose and galactose as the predominating structural units. The mean M(r) was determined by HP-GPC. The heteroglycans were tested for in vitro immunomodulating activities and showed mitogenic activity in rat spleen cell proliferation assay and stimulated IL-10 secretion. In rat peritoneal macrophages, the heteroglycans stimulated TNF-alpha secretion, but not IL-10 secretion. These results indicate that the polysaccharides influence cells of the immune system both from the innate and the adaptive systems.     

A GGCAGG motif in minisatellites affecting their germline instability

Mouse and human genomes contain hypervariable DNA regions consisting of tandem repeats of a short sequence referred to as minisatellites. This variation is thought to arise through processes such as unequal crossover or replication slippage. A mo-1 minisatellite probe comprising a 14-base pair repeat sequence reveals many polymorphic fragments even in DNA of BALB/c sublines. Oligonucleotide probes with single base substitution in the mo-1 have been synthesized and used for assessing sequence involved in generation of polymorphisms. The results indicate that the loci containing mo-1 homologues with mutation in the GGCAGG sequence are monomorphic despite the other mutants showing polymorphism. Reciprocally, locus-specific polymorphic clones, Pc-1 and Pc-2, have been isolated with hybridization to mo-1, and both are shown to contain repeated sequence comprising the GGCAGG sequence. They reveal high mutation rates of 8.8% and 3.3% per gamete, respectively. These results strongly suggest that the motif contributes to the germline instability of minisatellites.     

Regional variation of Puccinia coronata avenae in Australia and its implications for oat breeding

A virulence survey of Puccinia coronata avenae was conducted in Australia from 1977 to 1980 with a chosen series of resistant oat lines as a tester set, viz., Ascencao, Pc-38, Pc-39, Pc-45, Pc-48, Pc-50, Pc-55, Pc-56 and TAM 0–312. Isolates of the pathogen were classified by using these nine tester lines in combination with the International Differential Set. The survey area was divided into six zones (1) -Queensland; (2) - northern New South Wales; (3) - southern New South Wales; (4) - Victoria; (5) - South Australia and (6) - Western Australia. Virulence for Pc-38 was widespread throughout Australia and virulences or partial virulences for lines Pc-39, Pc-45, Pc-48 and Pc-55, although not as frequent, were common in Zones 1, 2 and 3. No isolates were found with virulences for oat lines Pc-56 and TAM 0–312, while virulences for Ascencao or Pc-50 were rare. The combination of virulences or partial virulences for lines Pc-39, Pc-45 and Pc-48 was relatively common only among samples from Zones 1, 2 and 3. The diversity of the pathogen population varied between zones. Isolates from Zones 1 to 6 comprised 16, 21, 9, 8, 5 and 4 different standard races and mean virulence values were 5-0, 3–8, 3-0, 2–7, 2–6, 3-1 and 3-0, respectively. The proportion of complex strains (virulence value 5 or higher) decreased from 48-5% (Zone 1) and 17-0% (Zone 2) to 1–7% (Zone 3), 6-3% (Zone 4), 0% (Zone 5) and 13-9% (Zone 6). Greater diversity of the pathogen population in Zones 1 and 2was indicated by a higher incidence of strains with virulence for one or more of the nine resistant oat tester lines.     

Lack of effects of atomic bomb radiation on genetic instability of tandem-repetitive elements in human germ cells.

In a pilot study to detect the potential effects of atomic bomb radiation on germ-line instability, we screened 64 children from 50 exposed families and 60 from 50 control families for mutations at six minisatellite loci by using Southern blot analysis with Pc-1, lambda TM-18, ChdTC-15, p lambda 3, lambda MS-1, and CEB-1 probes. In the exposed families, one or both parents received a radiation dose > 0.01 Sv. Among the 64 children, only one child had parents who were both exposed. Thus, of a total of 128 gametes that produced the 64 children, 65 gametes were derived from exposed parents and 63 were from unexposed parents, the latter being included in a group of 183 unexposed gametes used for calculating mutation rates. The average parental gonadal dose for the 65 gametes was 1.9 Sv. We detected a total of 28 mutations at the p lambda g3, lambda MS-1, and CEB-1 loci, but no mutations at the Pc-1, lambda TM-18, and ChdTC-15 loci. We detected 6 mutations in 390 alleles of the 65 exposed gametes and 22 mutations in 1098 alleles of the 183 gametes from the unexposed parents. The mean mutation rate per locus per gamete in these six minisatellite loci was 1.5% in the exposed parents and 2.0% in the unexposed parents. We observed no significant difference in mutation rates in the children of the exposed and the unexposed parents (P = .37, Fisher's exact probability test).     

Cement proteins of the tube-building polychaete Phragmatopoma californica

The mineralized tube of the sandcastle worm Phragmatopoma californica is made from exogenous mineral particles (sand, shell, etc.) glued together with a cement secreted from the "building organ" on the thorax of the worm. The glue is a cross-linked mixture of three highly polar proteins. The complete sequences of Pc-1 (18 kDa) and Pc-2 (21 kDa) were deduced from cDNAs derived from previously reported peptide sequences (Waite, J. H., Jensen, R., and Morse, D. E. (1992) Biochemistry 31, 5733-5738). Both proteins are basic (pI approximately 10) and exhibit Gly-rich peptide repeats. The consensus repeats in Pc-1 and -2 are VGGYGYGGKK (15 times), and HPAVXHKALGGYG (eight times), respectively, in which X denotes an intervening nonrepeated sequence and Y is modified to 3,4-dihydroxyphenyl-l-alanine (Dopa). The third protein, Pc-3, was deduced from the cement to be about 80 mol % phosphoserine/serine, and the cDNA was obtained by exploiting the presence of poly-serine repeats. Pc-3 consists of a family of at least seven variants with 60-90 mol % serine most of which is phosphorylated in the cement. Pc-1, -2, and -3 contain cysteine some of which reacts to form 5-S-cysteinyl-Dopa cross-links during the setting process.     

Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5′-flaps

The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3′–5′ exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol δ-exo⁻ to carry out strand displacement synthesis and discovered that it is regulated by the 5′-flaps in the DNA strand to be displaced. Under conditions where Pol δ carries out strand displacement synthesis, the presence of long 5′-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5′-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol δ with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol δ activities.     

Molecular activities of meiosis-specific proteins Hop2, Mnd1, and the Hop2-Mnd1 complex

The mouse Hop2 and Mnd1 proteins, which can form a stable heterodimeric complex, ensure the proper synapsis of homologous chromosomes in meiosis by acting in concert with Rad51 and Dmc1 to promote the strand invasion (D-loop formation) step of homologous recombination. Hop2 alone promotes D-loop formation, but Mnd1 and the Hop2-Mnd1 complex do not. Here we show that only the heterodimer complex, but not the individual proteins, can stimulate strand invasion by Dmc1. Furthermore, we demonstrate that the interaction with Mnd1 provokes changes in Hop2 that are responsible not only for abrogating the recombinase activity of Hop2 but also for generating a new molecular interface able to physically interact with and stimulate Dmc1. We also show that coiled-coil motifs in Hop2 and Mnd1 are essential for their interaction with each other and that a clearly delineated region near the COOH terminus of both proteins is necessary for both the DNA binding and single-strand annealing by the Hop-Mnd1 heterodimer. Finally, we describe a point mutation in Hop2 that dissociates its strand invasion activity from its ability to bind and anneal DNA.     

Generation of DNA double-strand breaks by two independent enzymatic activities in nuclear extracts

We have reported the existence in rat nuclear extracts of a specific cleavage activity on a DNA fragment containing the human minisatellite MsH42 region (minisatellite plus its flanking sequences). Here, we have developed a system to analyse the nature of the cleavage products from the MsH42 region generated by the nuclear extracts. Our results demonstrated the formation of DNA double-strand breaks (DSB) in the MsH42 region by two different enzymatic activities, and that their distribution along this fragment changes depending on the presence of Mg2+. In the assays with Mg2+, the DSB were located in the minisatellite and its 3'-flanking region, showing preference for G-rich stretches. Oligonucleotide mutagenesis analysis confirmed that this enzymatic activity has a strong preference for G-tracts and that the recognition site is polarized towards the 3' end. Moreover, this activity cuts GC palindromes efficiently. In contrast, in the experiments without Mg2+, most DSB were mapped within the minisatellite flanking sequences. The analysis with oligonucleotides showed that G-tracts are recognized by this endonuclease activity, but with differences in the cleavage behaviour with respect to the reactions observed with Mg2+. The existence of two separate activities (Mg2+-dependent and Mg2+-independent) for the production of DSB was confirmed by analysing the effect of EGTA, N-ethyl maleimide, ionic strength, and by preincubations of the nuclear extracts at different temperatures. The tissue distribution of both DSB-producing activities was also different. The in vitro system used in the present work may be a useful tool for studying the formation of DSB and for investigation of the mechanisms of DNA repair.     

Human intra-intronic minisatellite UPS29 associated with neurological diseases regulates reporter gene EGFP expression depending on cell type

Earlier, it was established that polymorphism of minisatellite UPS29 located in one of introns of human gene CENTB5 (ACAP3) was associated with Parkinson's disease and epilepsy. The main aim of this work was to elucidate if that minisatellite could regulate reporter gene activity, and if such activity was tissue (cell)-specific. To this end there was used transient transfection of HeLa cells, mouse embryonal carcinoma line F9, and rat astrocytes cultures with plasmides which contained reporter gene EGFP under eukaryotic promoter ROSA26 and different allelles of minisatellite UPS29. It was found that UPS29 possessed enhancer-like activity in neuronal type cells.     

Rational proteomics of PKD1. I. Modeling the three dimensional structure and ligand specificity of the C_lectin binding domain of Polycystin-1.

Polycystin-1 (Pc-1) is the 4303 amino acid multi-domain glycoprotein product of the polycystic kidney disease-1 (PKD1) gene. Mutations in this gene are implicated in 85% of cases of human autosomal dominant polycystic disease. Although the biochemistry of Pc-1 has been extensively studied its three dimensional structure has yet to be determined. We are combining bioinformatics, computational and biochemical data to model the 3D structure and function of individual domains of Pc-1. A three dimensional model of the C-type lectin domain (CLD) of Pc-1 (sequence region 405–534) complexed with galactose (Gal) and a calcium ion (Ca⁺²) has been developed (the coordinates are available on request, e-mail: pletnev@hwi.buffalo.edu). The model has α/β structural organization. It is composed of eight β strands and three α helices, and includes three disulfide bridges. It is consistent with the observed Ca⁺² dependence of sugar binding to CLD and identifies the amino acid side chains (E499, H501, E506, N518, T519 and D520) that are likely to bind the ligand. The model provides a reliable basis upon which to map functionally important residues using mutagenic experiments and to refine our knowledge about a preferred sugar ligand and the functional role of the CLD in polycystin-1. Figure Carbohydrate binding site with bound galactose and calcium ion inC-lectin binding domain of polycystin-1