G. Goellner - Academia.edu (original) (raw)
Papers by G. Goellner
Cell, 1995
We show that repeating units from all reported disease genes are capable of forming hairpins of c... more We show that repeating units from all reported disease genes are capable of forming hairpins of common structure and threshold stability. The threshold stability is roughly-50 kcal per hairpin and is influenced by the flanking sequence of the gene. Hairpin stability has two components, sequence and length; only DNA of select sequences and the correct length can form hairpins of threshold energy. There is a correlation among the ability to form hairpins of threshold stability, the sequence selectivity of expansion, and the length dependence of expansion. Additionally, hairpin formation provides a potential structural basis for the constancy of the CCG region of the Huntington's disease gene in individuals and explains the stabilizing effects of AGG interruptions in FMRf alleles.
Human Molecular Genetics, 2005
Huntington's disease (HD) is one of a group of neurodegenerative disorders caused by the patholog... more Huntington's disease (HD) is one of a group of neurodegenerative disorders caused by the pathological expansion of a glutamine tract. A hallmark of these so-called polyglutamine diseases is the presence of ubiquitylated inclusion bodies, which sequester various components of the 19S and 20S proteasomes. In addition, the ubiquitin-proteasome system (UPS) has been shown to be severely impaired in vitro in cells overexpressing mutant huntingtin. Thus, because of its fundamental housekeeping function, impairment of the UPS in neurons could contribute to neurotoxicity. We have recently proposed that the proteasome activator REGg could contribute to UPS impairment in polyglutamine diseases by suppressing the proteasomal catalytic sites responsible for cleaving Gln-Gln bonds. Capping of proteasomes with REGg could therefore contribute to a potential 'clogging' of the proteasome by pathogenic polyglutamines. We show here that genetic reduction of REGg has no effect on the well-defined neurological phenotype of R6/2 HD mice and does not affect inclusion body formation in the R6/2 brain. Surprisingly, we observe increased proteasomal 'chymotrypsin-like' activity in 13-week-old R6/2 brains relative to non-R6/2, irrespective of REGg levels. However, assays of 26S proteasome activity in mouse brain extracts reveal no difference in proteolytic activity regardless of R6/2 or REGg genotype. These findings suggest that REGg is not a viable therapeutic target in polyglutamine disease and that overall proteasome function is not impaired by trapped mutant polyglutamine in R6/2 mice.
Two recent lines of evidence raise the possibility that instability in germ-line or somatic cells... more Two recent lines of evidence raise the possibility that instability in germ-line or somatic cells arises by a common mechanism that involves defective mismatch repair. Mutations in mismatch-repair proteins are known to cause instability in hereditary nonpolyposis colorectal cancer, instability that is physically similar to germ-line instability observed in Huntington disease (HD). Furthermore, both germ-line and somatic-cell instability are likely to be mitotic defects, the former occurring early in embryogenesis. To test the hypothesis that defective repair is a common prerequisite for instability, we have utilized two disease groups that represent different instability "conditions." Germ-line instability within simple tandem repeats (STR) at 10 loci in 29 HD families were compared with somatic instability at the same loci in 26 colon cancer (CC) patients with identified or suspected defects in mismatch-repair enzymes. HD is known to be caused by expansion within the CAG ...
American journal of human genetics, 1997
Two recent lines of evidence raise the possibility that instability in germ-line or somatic cells... more Two recent lines of evidence raise the possibility that instability in germ-line or somatic cells arises by a common mechanism that involves defective mismatch repair. Mutations in mismatch-repair proteins are known to cause instability in hereditary nonpolyposis colorectal cancer, instability that is physically similar to germ-line instability observed in Huntington disease (HD). Furthermore, both germ-line and somatic-cell instability are likely to be mitotic defects, the former occurring early in embryogenesis. To test the hypothesis that defective repair is a common prerequisite for instability, we have utilized two disease groups that represent different instability "conditions." Germ-line instability within simple tandem repeats (STR) at 10 loci in 29 HD families were compared with somatic instability at the same loci in 26 colon cancer (CC) patients with identified or suspected defects in mismatch-repair enzymes. HD is known to be caused by expansion within the CAG ...
The EMBO Journal, 2000
Recombination induced by double-strand breaks (DSBs) in yeast leads to a higher proportion of exp... more Recombination induced by double-strand breaks (DSBs) in yeast leads to a higher proportion of expansions to contractions than does replication-associated tract length changes. Expansions are apparently dependent on the property of the repeat array to form hairpins, since DSB repair of a CAA 87 repeat induces only contractions of the repeat sequence. DSB-repair ef®ciency is reduced by 40% when DNA synthesis must traverse a CAG 98 array, as compared with a CAA 87 array. These data indicate that repairassociated DNA synthesis is inhibited by secondary structures formed by CAG 98 and that these structures promote repeat expansions during DSB repair. Overexpression of Mre11p or Rad50p suppresses the inhibition of DSB repair by CAG 98 and signi®cantly increases the average size of expansions found at the recipient locus. Both effects are dependent on the integrity of the Mre11p±Rad50p±Xrs2p complex. The Mre11 complex thus appears to be directly involved in removing CAG or CTG hairpins that arise frequently during DNA synthesis accompanying gene conversion of these trinucleotide repeats.
Molecular Cell, 1998
We show that GAA instability in Friedreich's Ataxia is a DNA-directed mutation caused by ... more We show that GAA instability in Friedreich's Ataxia is a DNA-directed mutation caused by improper DNA structure at the repeat region. Unlike CAG or CGG repeats, which form hairpins, GAA repeats form a YRY triple helix containing non-Watson-Crick pairs. As with hairpins, triplex mediates intergenerational instability in 96% of transmissions. In families with Friedreich's Ataxia, the only recessive trinucleotide disease, GAA instability is not a function of the number of long alleles, ruling out homologous recombination or gene conversion as a major mechanism. The similarity of mutation pattern among triple repeat-related diseases indicates that all trinucleotide instability occurs by a common, intraallelic mechanism that depends on DNA structure. Secondary structure mediates instability by creating strong polymerase pause sites at or within the repeats, facilitating slippage or sister chromatid exchange.
Journal of Neurochemistry, 2001
Precise regulation of intracellular Ca 21 concentration ([Ca 21 ] i ) is achieved by the coordina... more Precise regulation of intracellular Ca 21 concentration ([Ca 21 ] i ) is achieved by the coordinated function of Ca 21 channels and Ca 21 buffers. Neuronal differentiation induces up-regulation of Ca 21 channels. However, little is known about the effects of differentiation on the expression of the plasma membrane Ca 21 -ATPase (PMCA), the principal Ca 21 extrusion mechanism in neurons. In this study, we examined the regulation of PMCA expression during differentiation of the human neuroblastoma cell line IMR-32. [Ca 21 ] i was monitored in single cells using indo-1 micro¯uorimetry. When the Ca 21 -ATPase of the endoplasmic reticulum was blocked by cyclopiazonic acid, [Ca 21 ] i recovery after small depolarization-induced Ca 21 loads was governed primarily by PMCAs. [Ca 21 ] i returned to baseline by a process described by a monoexponential function in undifferentiated cells (t 52^4 s; n 25).
Journal of Biological Chemistry, 2004
In addition to its thirty or so core subunits, a number of accessory proteins associate with the ... more In addition to its thirty or so core subunits, a number of accessory proteins associate with the 26 S proteasome presumably to assist in substrate degradation or to localize the enzyme within cells. Among these proteins is ecm29p, a 200-kDa yeast protein that contains numerous HEAT repeats as well as a putative VHS domain. Higher eukaryotes possess a well conserved homolog of yeast ecm29p, and we produced antibodies to three peptides in the human Ecm29 sequence. The antibodies show that Ecm29 is present exclusively on 26 S proteasomes in HeLa cells and that Ecm29 levels vary markedly among mouse organs. Confocal immunofluorescence microscopy localizes Ecm29 to the centrosome and a subset of secretory compartments including endosomes, the ER and the ERGIC. Ecm29 is up-regulated 2-3-fold in toxinresistant mutant CHO cells exhibiting increased rates of ER-associated degradation. Based on these results we propose that Ecm29 serves to couple the 26 S proteasome to secretory compartments engaged in quality control and to other sites of enhanced proteolysis.
Journal of Biological Chemistry, 2008
Small insertions and deletions of trinucleotide repeats (TNRs) can occur by polymerase slippage a... more Small insertions and deletions of trinucleotide repeats (TNRs) can occur by polymerase slippage and hairpin formation on either template or newly synthesized strands during replication. Although not predicted by a slippage model, deletions occur preferentially when 5'-CTG is in the lagging strand template and are highly favored over insertion events in rapidly replicating cells. The mechanism for the deletion bias and the orientation dependence of TNR instability is poorly understood. We report here that there is an orientation-dependent impediment to polymerase progression on 5'-CAG and 5'-CTG repeats that can be relieved by the binding of single-stranded DNA-binding protein. The block depends on the primary sequence of the TNR but does not correlate with the thermodynamic stability of hairpins. The orientation-dependent block of polymerase passage is the strongest when 5'-CAG is the template. We propose a "template-push" model in which the slow speed of DNA polymerase across the 5'-CAG leading strand template creates a threat to helicase-polymerase coupling. To prevent uncoupling, the TNR template is pushed out and by-passed. Hairpins do not cause the block, but appear to occur as a consequence of polymerase pass-over.
Biochemistry and Cell Biology, 2001
The mechanism of DNA expansion is not well understood. Recent evidence from genetic, in vivo, and... more The mechanism of DNA expansion is not well understood. Recent evidence from genetic, in vivo, and in vitro studies has suggested a link between the formation of alternative DNA secondary structures by trinucleotide repeat tracts and their propensity to undergo expansion. This review will focus on structural features and the mechanism of expansion relevant to human disease.
Cell, 1995
We show that repeating units from all reported disease genes are capable of forming hairpins of c... more We show that repeating units from all reported disease genes are capable of forming hairpins of common structure and threshold stability. The threshold stability is roughly -50 kcal per hairpin and is influenced by the flanking sequence of the gene. Hairpin stability has two components, sequence and length; only DNA of select sequences and the correct length can form hairpins of threshold energy. There is a correlation among the ability to form hairpins of threshold stability, the sequence selectivity of expansion, and the length dependence of expansion. Additionally, hairpin formation provides a potential structural basis for the constancy of the CCG region of the Huntington's disease gene in individuals and explains the stabilizing effects of AGG interruptions in FMRf alleles.
Cell, 1995
We show that repeating units from all reported disease genes are capable of forming hairpins of c... more We show that repeating units from all reported disease genes are capable of forming hairpins of common structure and threshold stability. The threshold stability is roughly-50 kcal per hairpin and is influenced by the flanking sequence of the gene. Hairpin stability has two components, sequence and length; only DNA of select sequences and the correct length can form hairpins of threshold energy. There is a correlation among the ability to form hairpins of threshold stability, the sequence selectivity of expansion, and the length dependence of expansion. Additionally, hairpin formation provides a potential structural basis for the constancy of the CCG region of the Huntington's disease gene in individuals and explains the stabilizing effects of AGG interruptions in FMRf alleles.
Human Molecular Genetics, 2005
Huntington's disease (HD) is one of a group of neurodegenerative disorders caused by the patholog... more Huntington's disease (HD) is one of a group of neurodegenerative disorders caused by the pathological expansion of a glutamine tract. A hallmark of these so-called polyglutamine diseases is the presence of ubiquitylated inclusion bodies, which sequester various components of the 19S and 20S proteasomes. In addition, the ubiquitin-proteasome system (UPS) has been shown to be severely impaired in vitro in cells overexpressing mutant huntingtin. Thus, because of its fundamental housekeeping function, impairment of the UPS in neurons could contribute to neurotoxicity. We have recently proposed that the proteasome activator REGg could contribute to UPS impairment in polyglutamine diseases by suppressing the proteasomal catalytic sites responsible for cleaving Gln-Gln bonds. Capping of proteasomes with REGg could therefore contribute to a potential 'clogging' of the proteasome by pathogenic polyglutamines. We show here that genetic reduction of REGg has no effect on the well-defined neurological phenotype of R6/2 HD mice and does not affect inclusion body formation in the R6/2 brain. Surprisingly, we observe increased proteasomal 'chymotrypsin-like' activity in 13-week-old R6/2 brains relative to non-R6/2, irrespective of REGg levels. However, assays of 26S proteasome activity in mouse brain extracts reveal no difference in proteolytic activity regardless of R6/2 or REGg genotype. These findings suggest that REGg is not a viable therapeutic target in polyglutamine disease and that overall proteasome function is not impaired by trapped mutant polyglutamine in R6/2 mice.
Two recent lines of evidence raise the possibility that instability in germ-line or somatic cells... more Two recent lines of evidence raise the possibility that instability in germ-line or somatic cells arises by a common mechanism that involves defective mismatch repair. Mutations in mismatch-repair proteins are known to cause instability in hereditary nonpolyposis colorectal cancer, instability that is physically similar to germ-line instability observed in Huntington disease (HD). Furthermore, both germ-line and somatic-cell instability are likely to be mitotic defects, the former occurring early in embryogenesis. To test the hypothesis that defective repair is a common prerequisite for instability, we have utilized two disease groups that represent different instability "conditions." Germ-line instability within simple tandem repeats (STR) at 10 loci in 29 HD families were compared with somatic instability at the same loci in 26 colon cancer (CC) patients with identified or suspected defects in mismatch-repair enzymes. HD is known to be caused by expansion within the CAG ...
American journal of human genetics, 1997
Two recent lines of evidence raise the possibility that instability in germ-line or somatic cells... more Two recent lines of evidence raise the possibility that instability in germ-line or somatic cells arises by a common mechanism that involves defective mismatch repair. Mutations in mismatch-repair proteins are known to cause instability in hereditary nonpolyposis colorectal cancer, instability that is physically similar to germ-line instability observed in Huntington disease (HD). Furthermore, both germ-line and somatic-cell instability are likely to be mitotic defects, the former occurring early in embryogenesis. To test the hypothesis that defective repair is a common prerequisite for instability, we have utilized two disease groups that represent different instability "conditions." Germ-line instability within simple tandem repeats (STR) at 10 loci in 29 HD families were compared with somatic instability at the same loci in 26 colon cancer (CC) patients with identified or suspected defects in mismatch-repair enzymes. HD is known to be caused by expansion within the CAG ...
The EMBO Journal, 2000
Recombination induced by double-strand breaks (DSBs) in yeast leads to a higher proportion of exp... more Recombination induced by double-strand breaks (DSBs) in yeast leads to a higher proportion of expansions to contractions than does replication-associated tract length changes. Expansions are apparently dependent on the property of the repeat array to form hairpins, since DSB repair of a CAA 87 repeat induces only contractions of the repeat sequence. DSB-repair ef®ciency is reduced by 40% when DNA synthesis must traverse a CAG 98 array, as compared with a CAA 87 array. These data indicate that repairassociated DNA synthesis is inhibited by secondary structures formed by CAG 98 and that these structures promote repeat expansions during DSB repair. Overexpression of Mre11p or Rad50p suppresses the inhibition of DSB repair by CAG 98 and signi®cantly increases the average size of expansions found at the recipient locus. Both effects are dependent on the integrity of the Mre11p±Rad50p±Xrs2p complex. The Mre11 complex thus appears to be directly involved in removing CAG or CTG hairpins that arise frequently during DNA synthesis accompanying gene conversion of these trinucleotide repeats.
Molecular Cell, 1998
We show that GAA instability in Friedreich's Ataxia is a DNA-directed mutation caused by ... more We show that GAA instability in Friedreich's Ataxia is a DNA-directed mutation caused by improper DNA structure at the repeat region. Unlike CAG or CGG repeats, which form hairpins, GAA repeats form a YRY triple helix containing non-Watson-Crick pairs. As with hairpins, triplex mediates intergenerational instability in 96% of transmissions. In families with Friedreich's Ataxia, the only recessive trinucleotide disease, GAA instability is not a function of the number of long alleles, ruling out homologous recombination or gene conversion as a major mechanism. The similarity of mutation pattern among triple repeat-related diseases indicates that all trinucleotide instability occurs by a common, intraallelic mechanism that depends on DNA structure. Secondary structure mediates instability by creating strong polymerase pause sites at or within the repeats, facilitating slippage or sister chromatid exchange.
Journal of Neurochemistry, 2001
Precise regulation of intracellular Ca 21 concentration ([Ca 21 ] i ) is achieved by the coordina... more Precise regulation of intracellular Ca 21 concentration ([Ca 21 ] i ) is achieved by the coordinated function of Ca 21 channels and Ca 21 buffers. Neuronal differentiation induces up-regulation of Ca 21 channels. However, little is known about the effects of differentiation on the expression of the plasma membrane Ca 21 -ATPase (PMCA), the principal Ca 21 extrusion mechanism in neurons. In this study, we examined the regulation of PMCA expression during differentiation of the human neuroblastoma cell line IMR-32. [Ca 21 ] i was monitored in single cells using indo-1 micro¯uorimetry. When the Ca 21 -ATPase of the endoplasmic reticulum was blocked by cyclopiazonic acid, [Ca 21 ] i recovery after small depolarization-induced Ca 21 loads was governed primarily by PMCAs. [Ca 21 ] i returned to baseline by a process described by a monoexponential function in undifferentiated cells (t 52^4 s; n 25).
Journal of Biological Chemistry, 2004
In addition to its thirty or so core subunits, a number of accessory proteins associate with the ... more In addition to its thirty or so core subunits, a number of accessory proteins associate with the 26 S proteasome presumably to assist in substrate degradation or to localize the enzyme within cells. Among these proteins is ecm29p, a 200-kDa yeast protein that contains numerous HEAT repeats as well as a putative VHS domain. Higher eukaryotes possess a well conserved homolog of yeast ecm29p, and we produced antibodies to three peptides in the human Ecm29 sequence. The antibodies show that Ecm29 is present exclusively on 26 S proteasomes in HeLa cells and that Ecm29 levels vary markedly among mouse organs. Confocal immunofluorescence microscopy localizes Ecm29 to the centrosome and a subset of secretory compartments including endosomes, the ER and the ERGIC. Ecm29 is up-regulated 2-3-fold in toxinresistant mutant CHO cells exhibiting increased rates of ER-associated degradation. Based on these results we propose that Ecm29 serves to couple the 26 S proteasome to secretory compartments engaged in quality control and to other sites of enhanced proteolysis.
Journal of Biological Chemistry, 2008
Small insertions and deletions of trinucleotide repeats (TNRs) can occur by polymerase slippage a... more Small insertions and deletions of trinucleotide repeats (TNRs) can occur by polymerase slippage and hairpin formation on either template or newly synthesized strands during replication. Although not predicted by a slippage model, deletions occur preferentially when 5'-CTG is in the lagging strand template and are highly favored over insertion events in rapidly replicating cells. The mechanism for the deletion bias and the orientation dependence of TNR instability is poorly understood. We report here that there is an orientation-dependent impediment to polymerase progression on 5'-CAG and 5'-CTG repeats that can be relieved by the binding of single-stranded DNA-binding protein. The block depends on the primary sequence of the TNR but does not correlate with the thermodynamic stability of hairpins. The orientation-dependent block of polymerase passage is the strongest when 5'-CAG is the template. We propose a "template-push" model in which the slow speed of DNA polymerase across the 5'-CAG leading strand template creates a threat to helicase-polymerase coupling. To prevent uncoupling, the TNR template is pushed out and by-passed. Hairpins do not cause the block, but appear to occur as a consequence of polymerase pass-over.
Biochemistry and Cell Biology, 2001
The mechanism of DNA expansion is not well understood. Recent evidence from genetic, in vivo, and... more The mechanism of DNA expansion is not well understood. Recent evidence from genetic, in vivo, and in vitro studies has suggested a link between the formation of alternative DNA secondary structures by trinucleotide repeat tracts and their propensity to undergo expansion. This review will focus on structural features and the mechanism of expansion relevant to human disease.
Cell, 1995
We show that repeating units from all reported disease genes are capable of forming hairpins of c... more We show that repeating units from all reported disease genes are capable of forming hairpins of common structure and threshold stability. The threshold stability is roughly -50 kcal per hairpin and is influenced by the flanking sequence of the gene. Hairpin stability has two components, sequence and length; only DNA of select sequences and the correct length can form hairpins of threshold energy. There is a correlation among the ability to form hairpins of threshold stability, the sequence selectivity of expansion, and the length dependence of expansion. Additionally, hairpin formation provides a potential structural basis for the constancy of the CCG region of the Huntington's disease gene in individuals and explains the stabilizing effects of AGG interruptions in FMRf alleles.