C. Freudenreich - Academia.edu (original) (raw)

Papers by C. Freudenreich

Proceedings of the National Academy of Sciences, 2011

Spinocerebellar ataxia 10 (SCA10) is an autosomal dominant disease caused by large-scale expansio... more Spinocerebellar ataxia 10 (SCA10) is an autosomal dominant disease caused by large-scale expansions of the (ATTCT) n repeat within an intron of the human ATXN10 gene. In contrast to other expandable repeats, this pentanucleotide repeat does not form stable intra- or interstranded DNA structures, being a DNA unwinding element instead. We analyzed the instability of the (ATTCT) n repeat in a yeast experimental system, where its expansions led to inactivation of the URA3 reporter gene. The inactivation was due to a dramatic decrease in the mRNA levels owing to premature transcription termination and RNA polyadenylation at the repeat. The rates of expansions strongly increased with the repeat's length, mimicking genetic anticipation in human pedigrees. A first round of genetic analysis showed that a functional TOF1 gene precludes, whereas a functional RAD5 gene promotes, expansions of the (ATTCT) n repeat. We hypothesize that repeat expansions could occur upon fortuitous template sw...

genome. yeast is dependent on its orientation in the Stability of a CTG/CAG trinucleotide repeat in

Cancer research, 1998

Various antitumor and antibacterial agents target type II DNA topoisomerases, stabilizing a cleav... more Various antitumor and antibacterial agents target type II DNA topoisomerases, stabilizing a cleaved DNA reaction intermediate and thereby converting topoisomerase into a cellular poison. Two 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)-resistant bacteriophage T4 topoisomerases have previously been characterized biochemically, and we have now determined the sequence of the causative mutations. In one case, a mutation (E457K) in a conserved domain of gp39 (ATPase subunit) causes resistance to antitumor agent m-AMSA but hypersensitivity to the quinolone oxolinic acid. In the second case, a combination of two amino acid substitutions (S79F and G269V) in gp52 (DNA-cleaving subunit) causes resistance to both m-AMSA and oxolinic acid. The S79F mutation is responsible for drug resistance, whereas the G269V mutation suppresses a topoisomerase deficiency caused by S79F. Surprisingly, the G269V mutation by itself causes a dramatic hypersensitivity to both inhibitors, defining a ...

BioTechniques, 1994

We have developed a method to produce a set of four duplex oligonucleotides, each with a differen... more We have developed a method to produce a set of four duplex oligonucleotides, each with a different labeled base at a given position, from one template-primer combination. The template oligonucleotide is synthesized with a mixture of all four bases at the position of interest, and the primer oligonucleotide hybridizes to the template at all bases 3' from the position of interest. Specifically labeled substrates are then produced by differential incorporation of each of the four labeled nucleotides in four separate reactions. This method is more cost-effective than synthesizing four separate duplex oligonucleotides with different base pairs at the position of interest. We have successfully used this method to test nucleotide substitutions at several positions of a DNA recognition site for the phage T4 type II DNA topoisomerase.

Human molecular …, 2000

An expansion to >200 CGG/CCG repeats (hereafter called CGG) in the 5′ ′ ′ ′ region of the FMR1 ge... more An expansion to >200 CGG/CCG repeats (hereafter called CGG) in the 5′ ′ ′ ′ region of the FMR1 gene causes fragile X syndrome, and this locus becomes a folatesensitive fragile site. We used Saccharomyces cerevisiae as a model system to study the stability and fragility of CGG repeats. Tracts of (CGG) 81 and (CGG) 160 were integrated onto a yeast chromosome in both orientations relative to the nearest replication origin. Tracts of this length are pre-mutation alleles in humans, with a high probability of expansion in future generations. The CGG tracts in yeast colonies showed a length-dependent instability with longer tracts being more prone to contraction than shorter tracts. In addition, there was an orientation bias for tract stability with tracts having fewer contractions when the CCG strand was the template for lagging strand synthesis. Expansions of the CGG tracts also occurred in an orientation-dependent manner, although at a lower frequency than contractions. To determine whether CGG tracts are fragile sites in yeast, the CGG tracts were flanked by direct repeats, and the rate of recombination between the repeats determined. Strains carrying the (CGG) 160 tract in either orientation had a large increase in their rate of recombination compared with a no-tract control strain. Because this increase was dependent on genes involved in doublestrand break repair, recombination was likely to be initiated by CGG tract-induced breakage between the direct repeats. The observation of orientationdependent instability and orientation-independent fragility suggests that at least some aspects of their underlying mechanisms are different.

Methods in molecular biology (Clifton, N.J.), 2001

... Kenneth N. Kreuzer, Catherine H. Freudenreich, and Yves Pommier ... 3-azido-AMSA absorbs maxi... more ... Kenneth N. Kreuzer, Catherine H. Freudenreich, and Yves Pommier ... 3-azido-AMSA absorbs maximally at about 350 nm [4]). 5. Reactive CPT derivatives: 7-ClMe-MDO-CPT and 7-Et-MDO-CPT were syn-thesized by Monroe E. Wall and Mansukh Wani (Research Triangle ...

Cancer research, Jan 15, 1998

Molecular and cellular biology, 1997

Trinucleotide repeat expansion is the causative mutation for a growing number of diseases includi... more Trinucleotide repeat expansion is the causative mutation for a growing number of diseases including myotonic dystrophy, Huntington's disease, and fragile X syndrome. A (CTG/CAG)130 tract cloned from a myotonic dystrophy patient was inserted in both orientations into the genome of Saccharomyces cerevisiae. This insertion was made either very close to the 5' end or very close to the 3' end of a URA3 transcription unit. Regardless of its orientation, no evidence was found for triplet-mediated transcriptional repression of the nearby gene. However, the stability of the tract correlated with its orientation on the chromosome. In one orientation, the (CTG/CAG)130 tract was very unstable and prone to deletions. In the other orientation, the tract was stable, with fewer deletions and two possible cases of expansion detected. Analysis of the direction of replication through the region showed that in the unstable orientation the CTG tract was on the lagging-strand template and tha...

The EMBO journal, 1993

We have analyzed the DNA sequence requirements for cleavage of a 30 bp oligonucleotide that conta... more We have analyzed the DNA sequence requirements for cleavage of a 30 bp oligonucleotide that contains a strong bacteriophage T4 type II topoisomerase site. A novel method was used to generate substrates with each of the four nucleotides at 10 positions surrounding the cleavage site, and mutant substrates were also prepared for the four internal positions of the staggered cleavage site. The substrates were tested for cleavage in the presence of several inhibitors that induce enzyme-mediated cleavage: four antitumor agents of different classes (an aminoacridine, a substituted anthraquinone, an ellipticine derivative and an epipodophyllotoxin) and one antibacterial quinolone. At eight nucleotide positions flanking the cleavage site, the same preferred bases were found regardless of which inhibitor was present. These preferred bases show dyad symmetry with respect to the cleavage site, indicating that both protomers of the topoisomerase homodimer interact with DNA in an analogous manner....

BioTechniques, 1994

Science, 1998

A B C Receptor 1 Beads (PS-1) Receptor 2 Beads (PS-2)

Proceedings of the National Academy of Sciences, 1994

Type H topolsomerases are the targets of several classes of chemotherapeutic agents that stabiliz... more Type H topolsomerases are the targets of several classes of chemotherapeutic agents that stabilize an int te of the catalytic cycle with the enzyme covalently Abbreviations: m-AMSA, 4'-(9-acridinylamino)methanesulfon-manisidide; 3-azido-AMSA, 4'-(3-azido-9-acridinylamino)methanesulfon-m-anisidide.

Nucleic Acids Research, 2012

DNA sequences that form secondary structures or bind protein complexes are known barriers to repl... more DNA sequences that form secondary structures or bind protein complexes are known barriers to replication and potential inducers of genome instability. In order to determine which helicases facilitate DNA replication across these barriers, we analyzed fork progression through them in wild-type and mutant yeast cells, using 2-dimensional gel-electrophoretic analysis of the replication intermediates. We show that the Srs2 protein facilitates replication of hairpin-forming CGG/CCG repeats and prevents chromosome fragility at the repeat, whereas it does not affect replication of G-quadruplex forming sequences or a protein-bound repeat. Srs2 helicase activity is required for hairpin unwinding and fork progression. Also, the PCNA binding domain of Srs2 is required for its in vivo role of replication through hairpins. In contrast, the absence of Sgs1 or Pif1 helicases did not inhibit replication through structural barriers, though Pif1 did facilitate replication of a telomeric protein barrier. Interestingly, replication through a protein barrier but not a DNA structure barrier was modulated by nucleotide pool levels, illuminating a different mechanism by which cells can regulate fork progression through protein-mediated stall sites. Our analyses reveal fundamental differences in the replication of DNA structural versus protein barriers, with Srs2 helicase activity exclusively required for fork progression through hairpin structures.

Molecular and Cellular Biology, 2003

Expanded TNR tracts are both unstable (changing in length) and fragile (displaying an increased p... more Expanded TNR tracts are both unstable (changing in length) and fragile (displaying an increased propensity to break). We have investigated the relationship between fidelity of lagging-strand replication and both stability and fragility of TNRs. We devised a new yeast artificial chromomosme (YAC)-based assay for chromosome breakage to analyze fragility of CAG/CTG tracts in mutants deficient for proteins involved in laggingstrand replication: Fen1/Rad27, an endo/exonuclease involved in Okazaki fragment maturation, the nuclease/ helicase Dna2, RNase HI, DNA ligase, polymerase ␦, and primase. We found that deletion of RAD27 caused a large increase in breakage of short and long CAG/CTG tracts, and defects in DNA ligase and primase increased breakage of long tracts. We also found a correlation between mutations that increase CAG/CTG tract breakage and those that increase repeat expansion. These results suggest that processes that generate strand breaks, such as faulty Okazaki fragment processing or DNA repair, are an important source of TNR expansions.

Molecular and Cellular Biology, 2004

Genetic analysis of Saccharomyces cerevisiae FEN1 (RAD27) also reveals its important role in prev... more Genetic analysis of Saccharomyces cerevisiae FEN1 (RAD27) also reveals its important role in preventing trinucleotide repeat (TNR) expansion. In humans such expansion is associated with neurodegenerative diseases. In vitro, FEN1 can inhibit TNR expansion by employing its endonuclease activity to compete with DNA ligase I. Here we employed two yeast FEN1 nuclease mutants, rad27-G67S and rad27-G240D, to further define the mechanism by which FEN1 prevents TNR expansion. Using a yeast artificial chromosome system that can detect both TNR instability and fragility, we demonstrate that the G240D but not the G67S mutation increases both the expansion and fragility of a CTG tract in vivo. In vitro, the G240D nuclease is proficient in cleaving a fixed nonrepeat double flap; however, it exhibits severely impaired cleavage of both nonrepeat and CTG-containing equilibrating flaps. In contrast, wild-type FEN1 and the G67S mutant exhibit more efficient cleavage on an equilibrating flap than on a fixed CTG flap. The degree of TNR expansion and the amount of chromosome fragility observed in the mutant strains correlate with the severity of defective flap cleavage in vitro. We present a model to explain how flap equilibration and the unique tracking mechanism of FEN1 can collaborate to remove TNR flaps and prevent repeat expansion.

Genetics, 2010

Trinucleotide repeats can form secondary structures, whose inappropriate repair or replication ca... more Trinucleotide repeats can form secondary structures, whose inappropriate repair or replication can lead to repeat expansions. There are multiple loci within the human genome where expansion of trinucleotide repeats leads to disease. Although it is known that expanded repeats accumulate double-strand breaks (DSBs), it is not known which DSB repair pathways act on such lesions and whether inaccurate DSB repair pathways contribute to repeat expansions. Using Saccharomyces cerevisiae, we found that CAG/CTG tracts of 70 or 155 repeats exhibited significantly elevated levels of breakage and expansions in strains lacking MRE11, implicating the Mre11/Rad50/Xrs2 complex in repairing lesions at structure-forming repeats. About two-thirds of the expansions that occurred in the absence of MRE11 were dependent on RAD52, implicating aberrant homologous recombination as a mechanism for generating expansions. Expansions were also elevated in a sae2 deletion background and these were not dependent on RAD52, supporting an additional role for Mre11 in facilitating Sae2-dependent hairpin processing at the repeat. Mre11 nuclease activity and Tel1-dependent checkpoint functions were largely dispensable for repeat maintenance. In addition, we found that intact homologous recombination and nonhomologous end-joining pathways of DSB repair are needed to prevent repeat fragility and that both pathways also protect against repeat instability. We conclude that failure of principal DSB repair pathways to repair breaks that occur within the repeats can result in the accumulation of atypical intermediates, whose aberrant resolution will then lead to CAG expansions, contractions, and repeat-mediated chromosomal fragility.

Cytogenetic and Genome Research, 2003

The trinucleotide repeats that expand to cause human disease form hairpin structures in vitro tha... more The trinucleotide repeats that expand to cause human disease form hairpin structures in vitro that are proposed to be the major source of their genetic instability in vivo. If a replication fork is a train speeding along a track of doublestranded DNA, the trinucleotide repeats are a hairpin curve in the track. Experiments have demonstrated that the train can become derailed at the hairpin curve, resulting in significant damage to the track. Repair of the track often results in contrac-tions and expansions of track length. In this review we introduce the in vitro evidence for why CTG/CAG and CCG/CGG repeats are inherently unstable and discuss how experiments in model organisms have implicated the replication, recombination and repair machinery as contributors to trinucleotide repeat instability in vivo.

Proceedings of the National Academy of Sciences, 2011

Spinocerebellar ataxia 10 (SCA10) is an autosomal dominant disease caused by large-scale expansio... more Spinocerebellar ataxia 10 (SCA10) is an autosomal dominant disease caused by large-scale expansions of the (ATTCT) n repeat within an intron of the human ATXN10 gene. In contrast to other expandable repeats, this pentanucleotide repeat does not form stable intra-or interstranded DNA structures, being a DNA unwinding element instead. We analyzed the instability of the (ATTCT) n repeat in a yeast experimental system, where its expansions led to inactivation of the URA3 reporter gene. The inactivation was due to a dramatic decrease in the mRNA levels owing to premature transcription termination and RNA polyadenylation at the repeat. The rates of expansions strongly increased with the repeat's length, mimicking genetic anticipation in human pedigrees. A first round of genetic analysis showed that a functional TOF1 gene precludes, whereas a functional RAD5 gene promotes, expansions of the (ATTCT) n repeat. We hypothesize that repeat expansions could occur upon fortuitous template switching during DNA replication. The rate of repeat contractions was elevated in the Tof1 knockout strain, but it was not affected by the RAD5 gene. Supporting the notion of replication irregularities, we found that (ATTCT) n repeats also cause length-dependent chromosomal fragility in yeast. Repeat-mediated fragility was also affected by the Tof1 and Rad5 proteins, being reduced in their absence.

Proceedings of the National Academy of Sciences, 2011

genome. yeast is dependent on its orientation in the Stability of a CTG/CAG trinucleotide repeat in

Cancer research, 1998

BioTechniques, 1994

Human molecular …, 2000

Methods in molecular biology (Clifton, N.J.), 2001

Cancer research, Jan 15, 1998

Molecular and cellular biology, 1997

The EMBO journal, 1993

BioTechniques, 1994

Science, 1998

A B C Receptor 1 Beads (PS-1) Receptor 2 Beads (PS-2)

Proceedings of the National Academy of Sciences, 1994

Nucleic Acids Research, 2012

Molecular and Cellular Biology, 2003

Molecular and Cellular Biology, 2004

Genetics, 2010

Cytogenetic and Genome Research, 2003

Proceedings of the National Academy of Sciences, 2011

Spinocerebellar ataxia 10 (SCA10) is an autosomal dominant disease caused by large-scale expansio... more Spinocerebellar ataxia 10 (SCA10) is an autosomal dominant disease caused by large-scale expansions of the (ATTCT) n repeat within an intron of the human ATXN10 gene. In contrast to other expandable repeats, this pentanucleotide repeat does not form stable intra-or interstranded DNA structures, being a DNA unwinding element instead. We analyzed the instability of the (ATTCT) n repeat in a yeast experimental system, where its expansions led to inactivation of the URA3 reporter gene. The inactivation was due to a dramatic decrease in the mRNA levels owing to premature transcription termination and RNA polyadenylation at the repeat. The rates of expansions strongly increased with the repeat's length, mimicking genetic anticipation in human pedigrees. A first round of genetic analysis showed that a functional TOF1 gene precludes, whereas a functional RAD5 gene promotes, expansions of the (ATTCT) n repeat. We hypothesize that repeat expansions could occur upon fortuitous template switching during DNA replication. The rate of repeat contractions was elevated in the Tof1 knockout strain, but it was not affected by the RAD5 gene. Supporting the notion of replication irregularities, we found that (ATTCT) n repeats also cause length-dependent chromosomal fragility in yeast. Repeat-mediated fragility was also affected by the Tof1 and Rad5 proteins, being reduced in their absence.