1986 — 1989 |
Leffak, Michael |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Replication Polarity of Eucaryotic Genes @ Wright State University
The replication of chromosomes and individual genes is under cell-specific and developmental control. The nonrandom activation of replication origins and the asymmetry of chromatin replication at the molecular level have engendered the hypothesis that the potential for transcription of a gene is determined by the origin used to replicate that gene in the immediately preceding cell cycle. A novel method for determining replication polarity (the run-off replication assay) will be employed to test this hypothesis by comparing the direction of replication of specific genes in different states of transcriptional activity. The replication polarity of the following genes will be examined: (a) the alpha- and beta-globin genes of mouse erythroleukemia cells before and after induction by DMSO, (b) the alpha- and beta-globin genes of chick embryo fibroblasts before and after activation by Rous sarcoma virus transformation, and (c) the translocated (active) and nontranslocated (inactive) alleles of the c-myc cellular transforming gene in human Burkitt lymphoma cells. Discovery of a correlation between gene activity and replication polarity in these oncogenically transformed cell types would provide insight into both the mechanisms and cellular structures involved in transcriptional control in normal and tumor cells. Future application of the run-off replication technique will allow isolation of bona fide replication origins based on in vivo activity. The run-off replication technique involves completion of in vivo initiated nascent DNA chains in vitro, in the presence of the density labeled nucleotide bromodeoxyuridine triphosphate. The polarity of replication through a given segment of DNA is deduced after resolution of restriction enzyme generated DNA fragments by isopychic centrifugation, gel electrophoresis and blot hybridization to radiolabeled probes; those fragments most enriched in the density label being the farthest from the origin of replication. Preliminary experiments with the run-off replication technique demonstrate the faithful elongation of in vivo initiated chains in vitro, the sensitivity of detection of this method, and suggest that a correlation between replication polarity and gene activity does exist; thus the inactive avian histone H5 gene replicates from a downstream origin while the active H5 gene, and the active or inactive alpha-globin genes, replicate from upstream origins. Replication from an upstream origin therefore may be a necessary, but not sufficient, condition for transcription.
|
1 |
1987 |
Leffak, Michael |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Cdna Clones Related to Retinal Function @ Wright State University
The retinal pigment epithelial (RPE) layer serves multiple functions in maintaining photoreceptor cell viability. Among these, none is more critical than the phagocytic removal of the shed tips of the rod outer segments (ROS). To study this essential process we will use a genetic model of retinal degeneration, the Royal College of Surgeons (RCS) retinal dystrophic (rdy) rat, in which phagocytosis is 90-95% deficient. The defect in the RCS rat is detected at the RPE cell plasma membrane, where ROS binding occurs but ingestion is impaired. Because any of several steps in phagocytosis may be aberrant, we propose to study the primary defect in dystrophic rat RPE using the methods of molecular genetics. We will employ both nucleic acid probes, which require no prior assumptions regarding the expression site of the rdy mutation, as well as anit-RPE plasma membrane antibodies. The specific aim of this proposal is to identify cDNA clones which can be used to produce probes for the characterization of differences between normal and dystrophic RPE cells at the level of gene structure, RNA synthesis and processing, and protein systhesis and localization. These clones will be identified by two approaches: (1) the differential screening of an RPE cell cDNA expression library cloned in lambda gtll, with sequences complementary to normal and dystrophic rat RPE mRNA, and (2) immunological screening of the CDNA library with selected monoclonal antibodies to RPE plasma membranes. By these methods we will attempt to isolate sequences whose expression is altered as a result of the rdy mutation. The RPE cDNA library is valuable not only for understanding the RCS rat model; it will also premit further study of changes in RPE function which accompany normal development and aging. Moreover, under appropriate cross-hybridization conditions we can use these clones to analyse human RPE cells for age-related changes and genetic disorders. Thus, our long range goals include applying these techniques to the human system, to ask whether RPE cell involvement is primary or secondary to the etiology of retinal dysfunction.
|
1 |
1988 |
Leffak, Michael |
S15Activity Code Description: Undocumented code - click on the grant title for more information. |
Small Instrumentation Program @ Wright State University
biomedical equipment purchase;
|
1 |
1996 — 1998 |
Leffak, Michael |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Human C Myc Gene Replication @ Wright State University
The broad goals of our work are to identify the structures which define a mammalian DNA replication origin, and to understand the mechanism of origin activation. The focus of our experiments is the replication origin of the human c-myc gene. The c-myc origin is one of a limited number of chromosomal origins identified in complex eucaryotes, and the only metazoan origin to initiate autonomous replication in vitro and in transfected cells at the chromosomal initiation sites. c-myc is an immediate early response gene, activated by many mitogenic pathways in normal cells. Multiple promoter and enhancer elements are located in the region of the c-myc replication origin, 5' to the c-myc gene. Depending on growth conditions the c-myc protein can stimulate DNA synthesis and cell division or promote apoptosis, whereas aberrant c-myc gene expression can contribute to oncogenesis. These observations suggest that the c-myc replication origin region may be an intersection point for cellular networks of transcription and growth control. As such, the c-myc origin may be a site of action of oncogenic viral or chemical pathogens. Understanding the function of DNA elements in the c-myc replication origin is likely therefore to give new insight into mechanisms which control the cell division in normal and pathological states. Three Specific Aims will test the hypothesis that the c-myc origin comprises start sites for DNA synthesis, and cis-acting origin elements which regulate replication initiation. c-myc origin mutants will be constructed which have site-specific deletions or DNA substitutions. Aim 1 will examine the replication of the c-myc origin constructs targeted to specific chromosomal integration sites by adeno-associated virus vectors or the yeast FLP recombines. To contrast the activity of the c-myc origin in plasmids and in the chromosome, Aim 2 will use these constructs to identify sequences which affect the ability of plasmids to replicate autonomously in 293S cell extracts, and in transfected HeLa cells. These Aims will test the relative efficiencies of the origin constructs, and map the locations of initiation events. Also in Aims 1 and 2, nuclease digestion will be used to probe the chromatin structure of the active and inactive origin constructs. Aim 3 will identify protein binding sites in elements which influence the activity of the c-myc replication origin. c-myc origin fragments identified by mutation as important for plasmid ARS activity or chromosomal origin activity, and supercoiled c-myc origin constructs, will be incubated with 293S cell extracts in vitro under replication initiation conditions. Specific protein:DNA complexes will be analyzed by electrophoretic mobility shift and DNase I footprinting.
|
1 |
1999 — 2003 |
Leffak, Michael |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Human C Myc Gene Replication Origin @ Wright State University
The process of DNA replication is the primary physiological target for anti-proliferative drugs used to treat cancer. The broad goal of our work is to characterize the major cell cycle regulated step in mammalian DNA replication, the activation of origins to initiate DNA synthesis. Our experiments focus on the human c-myc replication origin. Understand the function of DNA elements in the c-myc origin is likely to give new insight into the regulation of DNA metabolism by mechanisms that control cell division in normal and disease states. The c-myc origin is one of a limited number of chromosomal origins identified in metazoans, and the only origin to replicate autonomously in plasmids in transfected cells and in vitro at chromosomal initiation sites. To test the effects of mutations on c-myc origin activity in the chromosomes of intact cells, we have developed an innovative system based on the S. cerevisae FLP recombinase for the site-specific integration of DNA in human cells. This system is highly efficient, and reproducibly targets c-myc origin constructs with precision to specific genomic acceptor sites. The FLP recombinase system is extremely flexible in the range of constructs that can be tested in an in vivo chromosomal environment. Hence, the system is not limited to the analysis of DNA replication but is broadly applicable to the study of other aspects of DNA metabolism. Using the FLP system we will test the hypothesis that the c-myc origin compromises preferred start sites for DNA synthesis and that initiation at these sites depends on cis-acting replicator elements. In each of three Specific Aims a panel of c-myc origin constructs will be integrated at defined chromosomal acceptor sites and the structure and replication activity at those sites before and after integration of the wild type and mutated origins will be assessed. Aim 1 will creative progressive 5' or 3' deletions of the origin, and mutations in specific candidate replicator elements, for analysis of origin activity. Aim 2 will test directly whether c-myc origin activity or replication timing is affected by an active transcription unit or telomere position effects. Aim 3 will test whether replication timing is affected by an active transcription unit or telomere position affects. Aim 3 will test whether there are multiple preferred start sites for the initiation of DNA synthesis in the c-myc origin that are subservient to a cis-acting replicator. Origin activity and structure will be analyzed by competitive PCR, PCR mapping of nascent DNA strands, DNase digestion, chemical footprinting, and ligation-mediated PCR.
|
1 |
2005 — 2012 |
Leffak, Michael |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Analysis of the Human C-Myc Gene Replication Origin @ Wright State University
DESCRIPTION (provided by applicant): DNA is most susceptible to damage during the process of replication. Therefore, complex mechanisms have evolved to ensure the accurate spatial and temporal initiation of DNA synthesis at replication origins, and the faithful copying of DNA during replication fork progression. Errors in the components of the multiprotein replication initiation complex, or the inability of tumor suppressor proteins to resolve blocks to fork movement lead to rearrangements, losses or duplications of DNA, and result in numerous genetic disorders. Our laboratory identified the replication origin 5'to the human c-myc oncogene, and we have shown that the structure, protein binding and function of the core c-myc replicator as a replication origin are the same at its endogenous chromosomal site and at ectopic sites in the human genome. In this application, the c-myc replicator will be used as a model mammalian replication origin to study the DNA sequences and protein factors that enable origin activity. In addition, the c-myc replicator will be used to initiate the replication of naturally occurring disease-related repeated DNA sequences that are impediments to replication fork progress. The proteins involved in sensing and stabilizing stalled replication forks are responsible for the suppression of genome instability, cancer, and neurodegenerative diseases. Because mammalian chromatin imposes constraints on replication that may not be recapitulated in other model systems, this is an innovative approach that allows the characterization of multiple sequences and DNA stress response proteins affecting replication fork stability in a chromatin environment. All three Aims will use site-directed FLP recombinase-mediated cassette exchange (FLP-RMCE) to integrate the c-myc replicator and its modified constructs into a unique site in the human genome. Aim 1 will use deletion analysis to identify protein binding sites in the c-myc replicator by quantitative chromatin immunoprecipitation (ChIP), define the minimal c-myc replication origin by PCR quantitation (qPCR) of nascent DNA, and assess the effect of Gal4 recruitment of replication proteins to the origin by ChIP and qPCR of nascent DNA. Aim 2 will test the effects of origin location, orientation, repeat sequence composition, and the effects of fork stabilizing proteins on the expansion or contraction of (CTG7CAG)n and (ATTCT7AGAAT)n microsatellites during replication. In Aim 3, a naturally occurring asymmetric polypurine7polypyrimidine sequence derived from the human PKD1/TSC2 locus, which we have shown to form a natural stalled replication fork, will be replicated from the ectopic c-myc origin and the role of replisome, DNA stress response proteins, translocase and helicases in fork stabilization and restart will be assessed by siRNA knockdown, ChIP, and qPCR of nascent DNA. We anticipate that these studies will give novel insight into the processes of replication initiation, replication fork progression, and genome stabilization in a human chromosomal environment. PUBLIC HEALTH RELEVANCE: Inappropriate regulation of DNA replication initiation can lead to chromosome fragmentation, abnormal cell division and human disease. We have developed a novel system that mimics the genomic instability observed in cancers and neurodegenerative diseases. Thus, our studies are likely to provide fundamental new insight into the mechanisms of DNA replication, the origins of genetic diseases, and new models for disease treatment.
|
1 |
2009 — 2010 |
Leffak, Michael |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Role of the Dna Unwinding Element Binding Protein, Due-B, in Dna Replication @ Wright State University
The initiation of DNA replication is a critical control point of the cell cycle, since abnormal replication initiation leads to genome instability, cancer and other inherited diseases. Accurate control of initiation requires the ordered assembly of prereplication protein and preinitiation protein complexes at origins of replication. We have identified a novel DNA unwinding element binding protein, DUE-B, which binds specifically to the essential DUE of the human c-myc replication origin and other origins. DUE-B is necessary for normal entry into the DNA synthetic S phase of the cell cycle and for the initiation of replication. DUE-B functions after formation of the prereplication complex but before origin template unwinding, and is required to load the preinitiation complex proteins Cdc45 and TopBP1 at origins. DUE-B therefore shows strong similarity to the yeast Cdc45-loading protein Sld3, a key target of the S phase promoting cyclin- dependent kinases. We propose to test a model in which metazoan DUE-B is the functional homolog of Sld3. Adding significance to the role of DUE B in replication, we have found a strong correlation between DUE-B overexpression and ovarian cancer. Remarkably, DUE-B also shows a second personality;the structure of the N-terminal 75% of the protein has been strongly conserved during evolution, and displays aminoacyl-tRNA proofreading activities found in yeast, bacteria and archae. We will use HeLa cells and Xenopus egg extracts to address several mechanistic aspects of the model in which DUE-B regulates the binding of the helicase activator Cdc45 to form the replication preinitiation complex, and in which the DUE-B-dependent loading of Cdc45 is a target of the intra-S phase DNA damage checkpoint. Our major analytical techniques will be immunoblotting, chromatin immunoprecipitation, quantitative PCR, FRET, and immunofluorescence. In Aim 1 we will analyze the binding of DUE-B and preinitiation complex proteins to replication origins, and the effect of the intra-S phase DNA damage checkpoint on DUE-B function. Aim 2 will characterize the effects of targeted structural mutations in DUE-B on its binding to protein ligands and its activation of replication origins.
|
1 |
2012 — 2015 |
Leffak, Michael |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Second-Site Genetic Modifiers of Ctg/Cag Microsatellite Stability @ Wright State University
DESCRIPTION (provided by applicant): Genomic instability of simple DNA sequence repeats (DNA microsatellites) is the cause of more than 30 human neurological diseases. In the case of myotonic dystrophy type 1 (DM1), an autosomal dominant disease of skeletal muscle with multiple phenotypes in other organs, the molecular trigger of disease is an increase in the number of CTG/CAG trinucleotide repeats in the 3' UTR of the DMPK gene. Dramatically increased CTG/CAG copy numbers can occur intergenerationally, or more modest expansions can occur somatically throughout life and differ substantially between tissues. The extent of CTG/CAG expansion is linked to increased disease severity and earlier age of onset of symptoms, but the penetrance of DM1 varies widely. This suggests that other genetic loci (second site genes) significantly affect the stability of CTG/CAG repeats in trans. The goal of this project is to characterize second site genes that contribute to CTG/CAG microsatellite instability. We will take a candidate gene approach to carry out the Aims of this work: (i) to identify genes required for the instability of DMPK (CTG/CAG) repeats by shRNA knockdown, small pool PCR and PAGE; (ii) to determine the effect of gene knockdown on the time course and length dependence of CTG/CAG instability, and effects on other disease-related microsatellites; (iii) to identify DNA hairpin pathways of CTG/CAG instability in vivo. The long term goals of this work are to understand the mechanistic basis for human CTG/CAG microsatellite instability in vivo, and the correlation of second site gene expression levels with DM1 phenotypes. We have engineered human cultured cells in which different lengths of DMPK CTG/CAG microsatellite repeat DNA have been inserted at a unique chromosomal location; this model assay system mimics the CTG/CAG instability observed in DM1 patient cells. Importantly, we have shown that knockdown of second site genes in DNA metabolic pathways promotes CTG/CAG repeat instability. The first result of this project will be the compilation of a list of genes whose expression levels could be used to predict CTG/CAG instability in a family, or in specific tissues of a patient. We will also perform molecular characterization of the effect of second site gene knockdown on the formation of unstable DNA hairpin intermediates in vivo, the rate of instability, the instability of pre-mutation CTG/CAG repeats, and the effects of these genetic modifiers on the stability of other microsatellites. The clinical value of these risk factors would include the prognosis of sporadic symptoms that are difficult to predict by periodic screening, and the individualization of treatment regimens. Similar tests of gene expression levels are currently in use by more than 7500 physicians and 90,000 patients to predict chemotherapy benefit and disease recurrence in breast and colon cancer. Our data generated thus far using this assay system as a sensor of DNA metabolism show that the identification of genes involved in CTG/CAG repeat instability will give insight into the basic mechanisms of genome stability and microsatellite expansion in multiple neurodegenerative disorders. PUBLIC HEALTH RELEVANCE: Expansions of short repeated DNA microsatellite sequences in the DMPK gene result in myotonic muscular dystrophy type 1 (DM1). To gain insight into the mechanisms of microsatellite expansion in DM1 and other neurological diseases we have engineered human cells in which different lengths of DMPK microsatellite repeat DNA have been inserted at a unique chromosomal location and have shown that decreased expression of specific genes promotes DMPK microsatellite instability. Our work will result in the identification of genes that contribute to microsatellite expansion and may therefore have clinical prognostic value, in addition to revealing molecular mechanisms of microsatellite instability.
|
1 |
2017 — 2020 |
Leffak, Michael |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms of Replication-Dependent Microsatellite Instability in Human Disease @ Wright State University
Chromosome breaks are the most dangerous form of DNA damage because they result in multiple types of mutations and gross chromosome rearrangements. DNA is most sensitive to breakage during replication, when hard-to-replicate noncanonical DNA structures cause replication fork stalling. Noncanonical DNA structures are strongly implicated as endogenous sources of chromosome breaks and translocations leading to developmental defects and cancers, however, the mechanisms by which replication fork stalling causes DNA double strand breaks (DSBs) are not known. Despite significant analyses of DNA damage response proteins in global or single molecule studies where the sites of damage are not identified, the molecular mechanisms of replication-dependent DNA strand breakage and repair at specific sites in human cells are incompletely understood. To address this knowledge gap, we will study two types of natural replication barriers (CTG/CAG trinucleotide repeats and asymmetric purine-pyrimidine (Pu/Py) mirror repeats) integrated at an ectopic site in the human genome where their structure and effect on replication can be manipulated. We also examine several endogenous replication fork barriers that induce DSBs during DNA replication. We will use PCR, DNA sequencing, chromatin immunoprecipitation, mass spectrometry and flow cytometry to show (1) how polymerase stalling at noncanonical DNA structures causes DSBs, (2) how DNA repair proteins act to remodel stalled replication forks to restart synthesis, and (3) the mechanisms and genomic consequences of DSB recombination at structure-induced fork barriers. We will test the hypothesis that noncanonical DNA structures induce DSB by blocking the progress of DNA polymerases, promoting nuclease-sensitive fork regression, and inhibiting DNA end processing required for recombination. Conceptual advances from this work will include determination of the molecular mechanisms of DSB formation near specific stalled forks, biochemical analysis of replication fork reversal, and identification of how the processing of structure-induced DSB differs that of nuclease-induced `clean' DSB. Our long-term goal is to define the role of DNA structure-induced g e n o m e instability in human disease. Aim 1 will disclose the relationship between fork stalling and damage signaling, the biochemistry of fork reversal, the function of structure-specific endonucleases at stalled forks, and the impact of DNA secondary structure on fork resection and repair. Aim 2 will build on our demonstration that the Fanconi anemia type J protein (FANCJ) is essential for the maintenance of noncanonical DNA structures across the genome during replication stress, to determine the mechanisms of FANCJ dependent microsatellite stabilization. In Aim 3 we will characterize the genomic consequences of FANCJ deficiency. Our experiments will show how hard-to- replicate DNA sequences cause chromosome breaks and mutations that lead to genetic disease.
|
1 |