Biomedical subjects

J D Griffith

Publications and source records attributed to J D Griffith.

At least 19 recordsLinked to original sources

PubMed2001

T-loop assembly in vitro involves binding of TRF2 near the 3' telomeric overhang.

Mammalian telomeres contain a duplex TTAGGG-repeat tract terminating in a 3' single-stranded overhang. TRF2 protein has been implicated in remodeling telomeres into duplex lariats, termed t-loops, in vitro and t-loops have been isolated from cells in vivo. To examine the features of the telomeric DNA essential for TRF2-promoted looping, model templates containing a 500 bp double-stranded TTAGGG tract and ending in different single-stranded overhangs were constructed. As assayed by electron microscopy, looped molecules containing most of the telomeric tract are observed with TRF2 at the loop junction. A TTAGGG-3' overhang of at least six nucleotides is required for loop formation. Termini with 5' overhangs, blunt ends or 3' termini with non-telomeric sequences at the junction are deficient in loop formation. Addition of non-telomeric sequences to the distal portion of a 3' overhang beginning with TTAGGG repeats only modestly diminishes looping. TRF2 preferentially localizes to the junction between the duplex repeats and the single-stranded overhang. Based on these findings we suggest a model for the mechanism by which TRF2 remodels telomeres into t-loops.

DNA, Single-Stranded↗

PubMed2001

Mechanism of topology simplification by type II DNA topoisomerases.

Type II DNA topoisomerases actively reduce the fractions of knotted and catenated circular DNA below thermodynamic equilibrium values. To explain this surprising finding, we designed a model in which topoisomerases introduce a sharp bend in DNA. Because the enzymes have a specific orientation relative to the bend, they act like Maxwell's demon, providing unidirectional strand passage. Quantitative analysis of the model by computer simulations proved that it can explain much of the experimental data. The required sharp DNA bend was demonstrated by a greatly increased cyclization of short DNA fragments from topoisomerase binding and by direct visualization with electron microscopy.

Computer Simulation↗

PubMed2001

t-loops at trypanosome telomeres.

Mammalian telomeres form large duplex loops (t-loops) that may sequester chromosome ends by invasion of the 3' TTAGGG overhang into the duplex TTAGGG repeat array. Here we document t-loops in Trypanosoma brucei, a kinetoplastid protozoan with abundant telomeres due to the presence of many minichromosomes. These telomeres contained 10-20 kb duplex TTAGGG repeats and a 3' TTAGGG overhang. Electron microscopy of psoralen/UV cross-linked DNA revealed t-loops in enriched telomeric restriction fragments and at the ends of isolated minichromosomes. In mammals, t-loops are large (up to 25 kb), often comprising most of the telomere. Despite similar telomere lengths, trypanosome t-loops were much smaller (approximately 1 kb), indicating that t-loop sizes are regulated. Coating of non-cross-linked minichromosomes with Escherichia coli single-strand binding protein (SSB) often revealed 3' overhangs at both telomeres and several cross-linked minichromosomes had t-loops at both ends. These results suggest that t-loops and their prerequisite 3' tails can be formed on the products of both leading and lagging strand synthesis. We conclude that t-loops are a conserved feature of eukaryotic telomeres.

Animals↗

PubMed2001

Electron microscopic analysis supports a dual role for the mitochondrial telomere-binding protein of Candida parapsilosis.

Linear mitochondrial genomes exist in several yeast species which are closely related to yeast that harbor circular mitochondrial genomes. Several lines of evidence suggest that the conversion from one form to another occurred accidentally through a relatively simple mechanism. Previously, we (L.T. & J.N.) reported the identification of the first mitochondrial telomere-binding protein (mtTBP) that specifically binds a sequence derived from the extreme end of Candida parapsilosis linear mtDNA, and sequence analysis of the corresponding nuclear gene MTP1 revealed that mtTBP shares homology with several bacterial and mitochondrial single-stranded (ss) DNA-binding (SSB) proteins. In this study, the DNA-binding properties of mtTBP in vitro and in vivo were analyzed by electron microscopy (EM). When M13 ssDNA was used as a substrate, mtTBP exhibited similar DNA binding characteristics as human mitochondrial SSB: mtTBP formed protein globules along the DNA substrate, and the bound proteins were randomly distributed, indicating that the binding of mtTBP to M13 ssDNA is not highly cooperative. EM analysis demonstrated that mtTBP is able to recognize the 5' single-stranded telomeric overhangs in their natural context. Using isopycnic centrifugation of mitochondrial lysates of C. papsilosis we show that mtTBP is a structural part of mitochondrial nucleoids of C. parapsilosis and is predominantly bound to the mitochondrial telomeres. These data support a dual role of mtTBP in mitochondria of C. parapsilosis, serving both as a typical mitochondrial SSB and as a specific component of the mitochondrial telomeric chromatin.

Bacteriophage M13↗

PubMed2000

Analysis of the binding of p53 to DNAs containing mismatched and bulged bases.

The tumor suppressor protein p53 modulates cellular response to DNA damage by a variety of mechanisms that may include direct recognition of some forms of primary DNA damage. Linear 49-base pair duplex DNAs were constructed containing all possible single-base mismatches as well as a 3-cytosine bulge. Filter binding and gel retardation assays revealed that the affinity of p53 for a number of these lesions was equal to or greater than that of the human mismatch repair complex, hMSH2-hMSH6, under the same binding conditions. However, other mismatches including G/T, which is bound strongly by hMSH2-hMSH6, were poorly recognized by p53. The general order of affinity of p53 was greatest for a 3-cytosine bulge followed by A/G and C/C mismatches, then C/T and G/T mismatches, and finally all the other mismatches.

Base Pair Mismatch↗

PubMed2000

Extragenomic double-stranded DNA circles in yeast with linear mitochondrial genomes: potential involvement in telomere maintenance.

Although the typical mitochondrial DNA (mtDNA) is portrayed as a circular molecule, a large number of organisms contain linear mitochondrial genomes classified by their telomere structure. The class of mitochondrial telomeres identified in three yeast species, Candida parapsilosis, Pichia philodendra and Candida salmanticensis, is characterized by inverted terminal repeats each consisting of several tandemly repeating units and a 5' single-stranded extension. The molecular mechanisms of the origin, replication and maintenance of this type of mitochondrial telomere remain unknown. While studying the replication of linear mtDNA of C.parapsilosis by 2-D gel electrophoresis distinct DNA fragments composed solely of mitochondrial telomeric sequences were detected and their properties were suggestive of a circular conformation. Electron microscopic analysis of these DNAs revealed the presence of highly supertwisted circular molecules which could be relaxed by DNase I. The minicircles fell into distinct categories based on length, corresponding to n x 0.75 kb (n = 1-7). Similar results were obtained with two other yeast species (P.philodendra and C. salmanticensis) which possess analogous telomeric structure.

Candida↗

PubMed2000

Regulation of origin recognition complex conformation and ATPase activity: differential effects of single-stranded and double-stranded DNA binding.

The Saccharomyces cerevisiae origin recognition complex (ORC) is bound to origins of DNA replication throughout the cell cycle and directs the assembly of higher-order protein-DNA complexes during G(1). To examine the fate of ORC when origin DNA is unwound during replication initiation, we determined the effect of single-stranded DNA (ssDNA) on ORC. We show that ORC can bind ssDNA and that ORC bound to ssDNA is distinct from that bound to double-stranded origin DNA. ssDNA stimulated ORC ATPase activity, whereas double-stranded origin DNA inhibited the same activity. Electron microscopy studies revealed two alternative conformations of ORC: an extended conformation stabilized by origin DNA and a bent conformation stabilized by ssDNA. Therefore, ORC appears to exist in two distinct states with respect to its conformation and ATPase activity. Interestingly, the effect of ssDNA on these properties of ORC is correlated with ssDNA length. Since double-stranded origin DNA and ssDNA differentially stabilize these two forms of ORC, we propose that origin unwinding triggers a transition between these alternative states.

Adenosine Triphosphatases↗

PubMed2000

HMG I/Y regulates long-range enhancer-dependent transcription on DNA and chromatin by changes in DNA topology.

The nature of nuclear structures that are required to confer transcriptional regulation by distal enhancers is unknown. We show that long-range enhancer-dependent beta-globin transcription is achieved in vitro upon addition of the DNA architectural protein HMG I/Y to affinity-enriched holo RNA polymerase II complexes. In this system, HMG I/Y represses promoter activity in the absence of an associated enhancer and strongly activates transcription in the presence of a distal enhancer. Importantly, nucleosome formation is neither necessary for long-range enhancer regulation in vitro nor sufficient without the addition of HMG I/Y. Thus, the modulation of DNA structure by HMG I/Y is a critical regulator of long-range enhancer function on both DNA and chromatin-assembled genes. Electron microscopic analysis reveals that HMG I/Y binds cooperatively to preferred DNA sites to generate distinct looped structures in the presence or absence of the beta-globin enhancer. The formation of DNA topologies that enable distal enhancers to strongly regulate gene expression is an intrinsic property of HMG I/Y and naked DNA.

Base Sequence↗

PubMed2000

Contingency management in outpatient methadone treatment: a meta-analysis.

A meta-analysis was conducted on contingency management interventions in outpatient methadone treatment settings. The outcome measure of interest was drug use during treatment, as detected through urinalysis. The results confirm that contingency management is effective in reducing supplemental drug use for these patients. The analysis of behavioral interventions yielded an overall effect size (r) of 0.25 based on 30 studies. Significant moderators of outcomes included type of reinforcement provided, time to reinforcement delivery, the drug targeted for behavioral change, number of urine specimens collected per week, and type of subject assignment. These factors represent important considerations for reducing drug use during treatment.

Behavior Therapy↗

PubMed2000

Betabellins 15D and 16D, de Novo designed beta-sandwich proteins that have amyloidogenic properties.

The betabellin structure is a de novo designed beta-sandwich protein consisting of two 32-residue beta-sheets packed against one another by hydrophobic interactions. d-Amino acid residues are used to energetically favor formation of type-I' beta turns. Air oxidation of betabellin 15S (B15S) (HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, where p denotes d-Pro, h denotes d-His, and k denotes d-Lys) yields betabellin 15D (B15D), a 64-residue disulfide-bridged protein. The amino acid sequence of B15D contains a conformationally constrained d-Pro residue at the i + 1 position of each type-I' beta turn. To test whether d-Pro residues are necessary for folding at these positions, the six d-Pro residues of B15D are replaced by d-Ala residues in betabellin 16D (B16D). Previously, transmission electron microscopy showed that B15D forms unbranched, 35-A wide fibrils that associate into bundles in 5.0 mM 3-(N-morpholino)propanesulfonate and 250 mM NaCl at pH 7; under these conditions, B16D forms ribbon-like assemblies. The B15D fibrils resemble the protofilaments that constitute amyloid fibrils. The present studies show that both B15D and B16D have characteristics of amyloidogenic proteins: the unbranched fibrils and ribbons stained with Congo red and displayed a green birefringence, exhibited a cross-beta structure, and bound 1-anilino-8-naphthalenesulfonate. Thus, these de novo designed beta-sandwich proteins should provide useful models for studying the mechanism of amyloid protofilament formation and assembly into amyloid fibrils and for designing potential inhibitors of amyloidogenesis.

Amino Acid Sequence↗

PubMed2000

Analysis of the Okazaki fragment distributions along single long DNAs replicated by the bacteriophage T4 proteins.

Rolling circle replication from M13 DNA circles was previously reconstituted in vitro using purified factors encoded by bacteriophage T4. The products are duplex circles with linear tails >100 kb. When T4 DNA polymerase deficient in 3' to 5' exonuclease activity was employed, electron microscopy revealed short single-stranded DNA "flaps" along the replicated tails. This marked the beginning of each Okazaki fragment, allowing an analysis of the lengths of sequential Okazaki fragments on individual replicating molecules. DNAs containing runs of Okazaki fragments of similar length were found, but most showed large length variations over runs of six or more fragments reflecting the broad population distribution.

Bacteriophage M13↗

PubMed1999

Biophysical characterization of betabellin 16D: a beta-sandwich protein that forms narrow fibrils which associate into broad ribbons.

The betabellin structure is a de novo designed beta-sandwich protein consisting of two 32-residue beta sheets packed against one another by hydrophobic interactions. Betabellin 16S (B16S), a 32-residue peptide chain (HSLTAKIakLTFSIAahTYTCAVakYTAKVSH, where a is DAla, h is DHis, and k is DLys), did not have beta structure in water at pH 6.5. Air oxidation of B16S furnished betabellin 16D (B16D), a 64-residue disulfide-bridged two-chain protein, which also did not fold in water at pH 6.5. However, the extent of beta structure observed for B16D increased with pH and ionic strength of the solution and the B16D concentration as observed by circular dichroism spectropolarimetry. Transmission electron microscopy showed that B16D formed narrow fibrils that associated into broad ribbons in 5.0 mM Mops and 0.25 M NaCl at pH 6.9.

Circular Dichroism↗

PubMed1999

TRF1 binds a bipartite telomeric site with extreme spatial flexibility.

TRF1 is a key player in telomere length regulation. Because length control was proposed to depend on the architecture of telomeres, we studied how TRF1 binds telomeric TTAGGG repeat DNA and alters its conformation. Although the single Myb-type helix-turn-helix motif of a TRF1 monomer can interact with telomeric DNA, TRF1 predominantly binds as a homodimer. Systematic Evolution of Ligands by Exponential enrichment (SELEX) with dimeric TRF1 revealed a bipartite telomeric recognition site with extreme spatial variability. Optimal sites have two copies of a 5'-YTAGGGTTR-3' half-site positioned without constraint on distance or orientation. Analysis of binding affinities and DNase I footprinting showed that both half-sites are simultaneously contacted by the TRF1 dimer, and electron microscopy revealed looping of the intervening DNA. We propose that a flexible segment in TRF1 allows the two Myb domains of the homodimer to interact independently with variably positioned half-sites. This unusual DNA binding mode is directly relevant to the proposed architectural role of TRF1.

Base Sequence↗

PubMed1999

Analysis of DNA replication forks encountering a pyrimidine dimer in the template to the leading strand.

Electron microscopy (EM) was used to visualize intermediates of in vitro replication of closed circular DNA plasmids. Cell-free extracts were prepared from human cells that are proficient (IDH4, HeLa) or deficient (CTag) in bypass replication of pyrimidine dimers. The DNA substrate was either undamaged or contained a single cis, syn thymine dimer. This lesion was inserted 385 bp downstream from the center of the SV40 origin of replication and sited specifically in the template to the leading strand of the newly synthesized DNA. Products from 30 minute reactions were crosslinked with psoralen and UV, linearized with restriction enzymes and spread for EM visualization. Extended single-stranded DNA regions were detected in damaged molecules replicated by either bypass-proficient or deficient extracts. These regions could be coated with Escherichia coli single-stranded DNA binding protein. The length of duplex DNA from a unique restriction site to the single-stranded DNA region was that predicted from blockage of leading strand synthesis by the site-specific dimer. These results were confirmed by S1nuclease treatment of replication products linearized with single cutting restriction enzymes, followed by detection of the diagnostic fragments by gel electrophoresis. The absence of an extended single-stranded DNA region in replication forks that were clearly beyond the dimer was taken as evidence of bypass replication. These criteria were fulfilled in 17 % of the molecules replicated by the IDH4 extract.

Cell Line, Transformed↗

PubMed1999

Mammalian telomeres end in a large duplex loop.

Mammalian telomeres contain a duplex array of telomeric repeats bound to the telomeric repeat-binding factors TRF1 and TRF2. Inhibition of TRF2 results in immediate deprotection of chromosome ends, manifested by loss of the telomeric 3' overhang, activation of p53, and end-to-end chromosome fusions. Electron microscopy reported here demonstrated that TRF2 can remodel linear telomeric DNA into large duplex loops (t loops) in vitro. Electron microscopy analysis of psoralen cross-linked telomeric DNA purified from human and mouse cells revealed abundant large t loops with a size distribution consistent with their telomeric origin. Binding of TRF1 and single strand binding protein suggested that t loops are formed by invasion of the 3' telomeric overhang into the duplex telomeric repeat array. T loops may provide a general mechanism for the protection and replication of telomeres.

Animals↗

PubMed1999

Sticky DNA: self-association properties of long GAA.TTC repeats in R.R.Y triplex structures from Friedreich's ataxia.

A novel DNA structure, sticky DNA, is described for lengths of (GAA.TTC)n found in intron 1 of the frataxin gene of Friedreich's ataxia patients. Sticky DNA is formed by the association of two purine.purine.pyrimidine (R.R.Y) triplexes in negatively supercoiled plasmids at neutral pH. An excellent correlation was found between the lengths of (GAA.TTC) (> 59 repeats): first, in FRDA patients, second, required to inhibit transcription in vivo and in vitro, and third, required to adopt the sticky conformation. Fourth, (GAAGGA.TCCTTC)65, also found in intron 1, does not form sticky DNA, inhibit transcription, or associate with the disease. Hence, R.R.Y triplexes and/or sticky DNA may be involved in the etiology of FRDA.

DNA↗

PubMed1999

Control of meiotic recombination and gene expression in yeast by a simple repetitive DNA sequence that excludes nucleosomes.

Tandem repeats of the pentanucleotide 5'-CCGNN (where N indicates any base) were previously shown to exclude nucleosomes in vitro (Y. -H. Wang and J. D. Griffith, Proc. Natl. Acad. Sci. USA 93:8863-8867, 1996). To determine the in vivo effects of these sequences, we replaced the upstream regulatory sequences of the HIS4 gene of Saccharomyces cerevisiae with either 12 or 48 tandem copies of CCGNN. Both tracts activated HIS4 transcription. We found that (CCGNN)(12) tracts elevated meiotic recombination (hot spot activity), whereas the (CCGNN)(48) tract repressed recombination (cold spot activity). In addition, a "pure" tract of (CCGAT)(12) activated both transcription and meiotic recombination. We suggest that the cold spot activity of the (CCGNN)(48) tract is related to the phenomenon of the suppressive interactions of adjacent hot spots previously described in yeast (Q.-Q. Fan, F. Xu, and T. D. Petes, Mol. Cell. Biol. 15:1679-1688, 1995; Q.-Q. Fan, F. Xu, M. A. White, and T. D. Petes, Genetics 145:661-670, 1997; T.-C. Wu and M. Lichten, Genetics 140:55-66, 1995; L. Xu and N. Kleckner, EMBO J. 16:5115-5128, 1995).

Alcohol Oxidoreductases↗

PubMed1998

Human Hsp70 and Hsp40 chaperone proteins facilitate human papillomavirus-11 E1 protein binding to the origin and stimulate cell-free DNA replication.

Human papillomavirus replication initiator, the E1 helicase, binds weakly to the origin of DNA replication. Purified human chaperone proteins Hsp70 and Hsp40 (HDJ-1 and HDJ-2) independently and additively enhanced E1 binding to the origin. The interaction between E1 and Hsp70 was transient and required ATP hydrolysis, whereas Hsp40 bound to E1 directly and remained in the complex. A peptide of 20 residues spanning the HPD loop and helix II of the J domain of YDJ-1 also stimulated E1 binding to the origin, alone or in combination with Hsp70 or Hsp40. A mutated peptide (H34Q) had a reduced activity, while an adjacent or an overlapping peptide had no effect. Neither Hsp70 nor the J peptide altered the E1/DNA ratio in the complex. Electron microscopy showed that E1 mainly bound to DNA as a hexamer. In the presence of Hsp40, E1 primarily bound to DNA as a dihexamer. Preincubation of chaperones with viral E1 and template shortened the lag time and increased replication in a cell-free system. Since two helicases are essential for bidirectional replication of human papillomavirus DNA, these results demonstrate that, as in prokaryotes, chaperones play an important role in the assembly of preinitiation complexes on the origin.

Carrier Proteins↗