Search PubMed⌕ Search

Biomedical subjects

E C Friedberg

Publications and source records attributed to E C Friedberg.

At least 73 records · Page 4Linked to original sources
PubMed1997

Transfection enhancement in Bacillus subtilis displays features of a novel DNA repair pathway. I: DNA base and nucleolytic specificity.

Cells of Bacillus subtilis can enter a natural physiological state, termed competence, that is permissive for uptake of DNA from the surrounding medium. In the B. subtilis genetic system, transfection refers to uptake of isolated bacteriophage DNA by competent host cells, followed by intracellular processing that may ultimately lead to productive infection. Previous investigations have shown that transfecting DNA is usually far less infectious (on a molar basis) than is the DNA injected by phage particles; this result is apparently due to inactivating events suffered by transfecting DNA during its metabolism by competent cells. Earlier studies also demonstrated that, in some cases, the infectivity of transfecting DNA can be increased by ultraviolet (UV) irradiation of the competent cells prior to transfection, or by cotransfection of UV-irradiated heterologous DNAs; collectively, these phenomena have been termed transfection enhancement (TE). We propose here that some transfecting B. subtilis phage DNAs are attacked by a novel host DNA repair system, and that TE reflects inhibition of this by a competing substrate in UV-irradiated DNA. In support of this model, we show that UV-DNA cotransfection leads to a reduced rate of intracellular endonucleolytic breakdown of transfecting DNA. We also demonstrate that TE displays marked specificity of a kind frequently observed for repair enzymes. Thus, phages that contain hydroxymethyl uracil (HMU), but not thymine, in their genomes are susceptible to this process. In addition, we show that the photoproduct(s) in UV-irradiated DNA that produces TE by cotransfection is specific, and is not uracil, a pyrimidine dimer, thymine glycol, HMU, or a substrate for the E. coli thymine glycol DNA N-glycosylase. This photoproduct is derivable from thymine or HMU. The implications of these results are discussed.

Bacillus subtilis↗
PubMed1997

The RAD7, RAD16, and RAD23 genes of Saccharomyces cerevisiae: requirement for transcription-independent nucleotide excision repair in vitro and interactions between the gene products.

Nucleotide excision repair (NER) is a biochemical process required for the repair of many different types of DNA lesions. In the yeast Saccharomyces cerevisiae, the RAD7, RAD16, and RAD23 genes have been specifically implicated in NER of certain transcriptionally repressed loci and in the nontranscribed strand of transcriptionally active genes. We have used a cell-free system to study the roles of the Rad7, Rad16, and Rad23 proteins in NER. Transcription-independent NER of a plasmid substrate was defective in rad7, rad16, and rad23 mutant extracts. Complementation studies with a previously purified NER protein complex (nucleotide excision repairosome) indicate that Rad23 is a component of the repairosome, whereas Rad7 and Rad16 proteins were not found in this complex. Complementation studies with rad4, rad7, rad16, and rad23 mutant extracts suggest physical interactions among these proteins. This conclusion was confirmed by experiments using the yeast two-hybrid assay, which demonstrated the following pairwise interactions: Rad4 with Rad23, Rad4 with Rad7, and Rad7 with Rad16. Additionally, interaction between the Rad7 and Rad16 proteins was demonstrated in vitro. Our results show that Rad7, Rad16, and Rad23 are required for transcription-independent NER in vitro. This process may involve a unique protein complex which is distinct from the repairosome and which contains at least the Rad4, Rad7, and Rad16 proteins.

Adenosine Triphosphatases↗
PubMed1996

Synergistic interactions between XPC and p53 mutations in double-mutant mice: neural tube abnormalities and accelerated UV radiation-induced skin cancer.

The significance of DNA repair to human health has been well documented by studies on xeroderma pigmentosum (XP) patients, who suffer a dramatically increased risk of cancer in sun-exposed areas of their skin [1,2]. This autosomal recessive disorder has been directly associated with a defect in nucleotide excision-repair (NER) [1,2]. Like human XP individuals, mice carrying homozygous mutations in XP genes manifest a predisposition to skin carcinogenesis following exposure to ultraviolet (UV) radiation [3-5]. Recent studies have suggested that, in addition to roles in apoptosis [6] and cell-cycle checkpoint control [7] in response to DNA damage, p53 protein may modulate NER [8]. Mutations in the p53 gene have been observed in 50% of all human tumors [9] and have been implicated in both the early [10] and late [11] stages of skin cancer. To examine the consequences of a combined deficiency of the XPC and the p53 proteins in mice, we generated double-mutant animals. We document a spectrum of neural tube defects in XPC p53 mutant embryos. Additionally, we show that, following exposure to UV-B radiation, XPC p53 mutant mice have more severe solar keratosis and suffer accelerated skin cancer compared with XPC mutant mice that are wild-type with respect to p53.

Animals↗
PubMed1996

UV-induced ubiquitination of RNA polymerase II: a novel modification deficient in Cockayne syndrome cells.

Damage to actively transcribed DNA is preferentially repaired by the transcription-coupled repair (TCR) system. TCR requires RNA polymerase II (Pol II), but the mechanism by which repair enzymes preferentially recognize and repair DNA lesions on Pol II-transcribed genes is incompletely understood. Herein we demonstrate that a fraction of the large subunit of Pol II (Pol II LS) is ubiquitinated after exposing cells to UV-radiation or cisplatin but not several other DNA damaging agents. This novel covalent modification of Pol II LS occurs within 15 min of exposing cells to UV-radiation and persists for about 8-12 hr. Ubiquitinated Pol II LS is also phosphorylated on the C-terminal domain. UV-induced ubiquitination of Pol II LS is deficient in fibroblasts from individuals with two forms of Cockayne syndrome (CS-A and CS-B), a rare disorder in which TCR is disrupted. UV-induced ubiquitination of Pol II LS can be restored by introducing cDNA constructs encoding the CSA or CSB genes, respectively, into CS-A or CS-B fibroblasts. These results suggest that ubiquitination of Pol II LS plays a role in the recognition and/or repair of damage to actively transcribed genes. Alternatively, these findings may reflect a role played by the CSA and CSB gene products in transcription.

Cell Line↗
PubMed1996

A yeast whole cell extract supports nucleotide excision repair and RNA polymerase II transcription in vitro.

Nucleotide excision repair (NER) and RNA polymerase II transcription are cellular processes that require the transcription/NER factor TFIIH. We have developed a whole cell extract from the yeast Saccharomyces cerevisiae that simultaneously supports both NER and RNA polymerase II transcription of independent substrates. NER activity in the yeast whole cell extract was readily detected in the absence of further supplementation but was stimulated in the presence of overexpressed Rad2 protein. The repair of N-acetyl-2-aminofluorene (AAF)-damaged DNA was dependent on RAD genes required for NER and deficient repair in rad mutant extracts was complemented by mixing different mutant extracts or by purified Rad proteins. Both the NER and transcription activities were stimulated by 5% polyethylene glycol in the whole cell extracts. Transcription activity from the template pCYC1G- was not affected by the presence of uracil-containing or AAF-damaged pUC18 DNA, which was expected to result in base excision repair (BER) and NER, respectively. An in vitro condition was defined that supported simultaneous NER and transcription independently in different substrates in the yeast whole cell extracts.

2-Acetylaminofluorene↗
PubMed1996

Identification of functional domains within the RAD1.RAD10 repair and recombination endonuclease of Saccharomyces cerevisiae.

Saccharomyces cerevisiae rad1 and rad10 mutants are unable to carry out nucleotide excision repair and are also defective in a mitotic intrachromosomal recombination pathway. The products of these genes are subunits of an endonuclease which recognizes DNA duplex/single-strand junctions and specifically cleaves the 3' single-strand extension at or near the junction. It has been suggested that such junctions arise as a consequence of DNA lesion processing during nucleotide excision repair and the processing of double-strand breaks during intrachromosomal recombination. In this study we show that the RAD1 RAD10 complex also cleaves a more complex junction structure consisting of a duplex with a protruding 3' single-strand branch that resembles putative recombination intermediates in the RAD1 RAD10-mediated single-strand annealing pathway of mitotic recombination. Using monoclonal antibodies, we have identified two regions of RAD1 that are required for the cleavage of duplex/single-strand junctions. These reagents also inhibit nucleotide excision repair in vitro, confirming the essential role of the RAD1 RAD10 endonuclease in this pathway.

Animals↗
PubMed1996

Cloning and characterization of RAD17, a gene controlling cell cycle responses to DNA damage in Saccharomyces cerevisiae.

Mutants of the yeast Saccharomyces cerevisiae defective in the RAD17 gene are sensitive to ultraviolet (UV) and gamma radiation and manifest a defect in G2 arrest following radiation treatment. We have cloned the RAD17 gene by complementation of the UV sensitivity of a rad17-1 mutant and identified an ORF of 1.2 kb encoding a predicted gene product of 45.4 kDa with homology to the Schizosaccharomyces pombe rad1+ gene product and to Ustilago maydis Rec1, a known 3'->5'exonuclease. The RAD17 transcript is cell cycle regulated, with maximum steady-state levels during late G1. The rad17-1 mutation represents a missense mutation that maps to a conserved region of the gene. A rad17 disruption mutant grows normally and manifests levels of UV sensitivity similar that of the rad17-1 strain. As previously observed with other genes involved in G2 arrest (such as RAD9 and RAD24), RAD17 regulates radiation-induced G1 checkpoints at at least two possible arrest stages. One is equivalent to or upstream of START, the other at or downstream of the Cdc4 execution point. However, the temperature sensitivity of the cell cycle mutant dna1-1 (a G1 arrest mutant) is not influenced by inactivation of RAD17.

Amino Acid Sequence↗
PubMed1996

Interactions involving the human RNA polymerase II transcription/nucleotide excision repair complex TFIIH, the nucleotide excision repair protein XPG, and Cockayne syndrome group B (CSB) protein.

The human basal transcription factor TFIIH plays a central role in two distinct processes. TFIIH is an obligatory component of the RNA polymerase II (RNAP II) transcription initiation complex. Additionally, it is believed to be the core structure around which some if not all the components of the nucleotide excision repair (NER) machinery assemble to constitute a nucleotide excision repairosome. At least two of the subunits of TFIIH (XPB and XPD proteins) are implicated in the disease xeroderma pigmentosum (XP). We have exploited the availability of the cloned XPB, XPD, p62, p44, and p34 genes (all of which encode polypeptide subunits of TFIIH) to examine interactions between in vitro-translated polypeptides by co-immunoprecipitation. Additionally we have examined interactions between TFIIH components, the human NER protein XPG, and the CSB protein which is implicated in Cockayne syndrome (CS). Our analyses demonstrate that the XPB, XPD, p44, and p62 proteins interact with each other. XPG protein interacts with multiple subunits of TFIIH and with CSB protein.

Base Sequence↗
PubMed1996

Rodent complementation group 8 (ERCC8) corresponds to Cockayne syndrome complementation group A.

US31 is a UV-sensitive mutant cell line (rodent complementation group 8) derived from a mouse T cell line L5178Y. We analyzed removal kinetics for UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts in US31 cells using monoclonal antibodies against these photoproducts. While nearly all (6-4) photoproducts were repaired within 6 h after UV-irradiation, more than 70% of cyclobutane pyrimidine dimers remained unrepaired even 24 h after UV-irradiation. These kinetics resembled those of Cockayne syndrome (CS) cells. Since US31 cells had a low efficiency of cell fusion and transfection, which hampered both complementation tests and gene cloning, we constructed fibroblastic complementation group 8 cell line 6L1030 by fusion of US31 cells with X-irradiated normal mouse fibroblastic LTA cells. Complementation tests by cell fusion and transfection using 6L1030 cells revealed that rodent complementation group 8 corresponded to CS complementation group A.

Animals↗
PubMed1996

Cockayne syndrome--a primary defect in DNA repair, transcription, both or neither?

Cockayne syndrome is a rare autosomal recessive disease characterized by a complex clinical phenotype. Most Cockayne syndrome cells are hypersensitive to killing by ultraviolet radiation. This observation has prompted a wealth of studies on the DNA repair capacity of Cockayne syndrome cells in vitro. Many studies support the notion that such cells are defective in a DNA repair mode(s) that is transcription-dependent. However, it remains to be established that this is a primary molecular defect in Cockayne syndrome cells and that it explains the complex clinical phenotype associated with the disease. An alternative hypothesis is that Cockayne syndrome cells have a defect in transcription affecting the expression of certain genes, which is compatible with embryogenesis but not with normal post-natal development. Defective transcription may impair the normal processing of DNA damage during transcription-dependent repair.

Cockayne Syndrome↗
PubMed1996

DNA repair and transcription.

The transcription factor TFIIH continues to be a subject of interest. In addition to its function as a repair and transcription factor, TFIIH includes a cyclin-dependent kinase and a cyclin, which raises the possibility that nucleotide excision repair (NER), RNA polymerase II transcription and cell cycle control are connected. Progress in mechanistic studies of NER include the identification of dual incision activities operating on either side of base damage and the isolation of a repairosome supercomplex in yeast. Additionally, NER has been demonstrated in reconstituted human and yeast systems, both of which include TFIIH.

Cell Cycle↗
PubMed1996

The Saccharomyces cerevisiae Ku autoantigen homologue affects radiosensitivity only in the absence of homologous recombination.

In mammalian cells, all subunits of the DNA-dependent protein kinase (DNA-PK) have been implicated in the repair of DNA double-strand breaks and in V(D)J recombination. In the yeast Saccharomyces cerevisiae, we have examined the phenotype conferred by a deletion of HDF1, the putative homologue of the 70-kD subunit of the DNA-end binding Ku complex of DNA-PK. The yeast gene does not play a role in radiation-induced cell cycle checkpoint arrest in G1 and G2 or in hydroxyurea-induced checkpoint arrest in S. In cells competent for homologous recombination, we could not detect any sensitivity to ionizing radiation or to methyl methanesulfonate (MMS) conferred by a hdf1 deletion and indeed, the repair of DNA double-strand breaks was not impaired. However, if homologous recombination was disabled (rad52 mutant background), inactivation of HDF1 results in additional sensitization toward ionizing radiation and MMS. These results give further support to the notion that, in contrast to higher eukaryotic cells, homologous recombination is the favored pathway of double-strand break repair in yeast whereas other competing mechanisms such as the suggested pathway of DNA-PK-dependent direct break rejoining are only of minor importance.

Antigens, Nuclear↗
PubMed1996

A mutation in a Saccharomyces cerevisiae gene (RAD3) required for nucleotide excision repair and transcription increases the efficiency of mismatch correction.

RAD3 functions in DNA repair and transcription in Saccharomyces cerevisiae and particular rad3 alleles confer a mutator phenotype, possibly as a consequence of defective mismatch correction. We assessed the potential involvement of the Rad3 protein in mismatch correction by comparing heteroduplex repair in isogenic rad3-1 and wild-type strains. The rad3-1 allele increased the spontaneous mutation rate but did not prevent heteroduplex repair or bias its directionality. Instead, the efficiency of mismatch correction was enhanced in the rad3-1 strain. This surprising result prompted us to examine expression of yeast mismatch repair genes. We determined that MSH2, but not MLH1, is transcriptionally regulated during the cell-cycle like PMS1, and that rad3-1 does not increase the transcript levels for these genes in log phase cells. These observations suggest that the rad3-1 mutation gives rise to an enhanced efficiency of mismatch correction via a process that does not involve transcriptional regulation of mismatch repair. Interestingly, mismatch repair also was more efficient when error-editing by yeast DNA polymerase delta was eliminated. We discuss our results in relation to possible mechanisms that may link the rad3-1 mutation to mismatch correction efficiency.

Adenosine Triphosphatases↗
PubMed1996

The Saccharomyces cerevisiae MEC1 gene, which encodes a homolog of the human ATM gene product, is required for G1 arrest following radiation treatment.

The Saccharomyces cerevisiae gene MEC1 represents a structural homolog of the human gene ATM mutated in ataxia telangiectasia patients. Like human ataxia telangiectasia cell lines, mec1 mutants are defective in G2 and S-phase cell cycle checkpoints in response to radiation treatment. Here we show an additional defect in G1 arrest following treatment with UV light or gamma rays and map a defective arrest stage at or upstream of START in the yeast cell cycle.

Ataxia Telangiectasia↗
PubMed1996

Molecular cloning and characterization of Saccharomyces cerevisiae RAD28, the yeast homolog of the human Cockayne syndrome A (CSA) gene.

Cockayne syndrome patients exhibit severe developmental and neurological abnormalities. Cells derived from these patients are sensitive to killing by UV radiation and do not support the rapid repair of the transcribed strand of transcriptionally active genes observed in cells from normal individuals. We report the cloning of the Saccharomyces cerevisiae homolog of the Cockayne syndrome A (CSA) gene, which we designate as RAD28. A rad28 null mutant does not manifest increased sensitivity to killing by UV or gamma radiation or to methyl methanesulfonate. Additionally, the rate of repair of the transcribed and nontranscribed strands of the yeast RPB2 gene in the rad28 mutant is identical to that observed in wild-type cells following exposure to UV light. As previously shown for rad7 rad26 and rad16 rad26 double mutants, the rad28 null mutant shows slightly enhanced sensitivity to UV light in the presence of mutations in the RAD7 or RAD16 gene. Both rad28 and rad26 null mutants are hypermutable following exposure to UV light.

Adenosine Triphosphatases↗
PubMed1996

Relationships between DNA repair and transcription.

Multiple relationships have been noted between DNA repair and transcription in both prokaryotic and eukaryotic cells. First, in both prokaryotes and eukaryotes nucleotide excision repair of the template strand of transcriptionally active regions of the genome is faster than in the coding strand. In prokaryotes the biochemical basis for this kinetic difference appears to be related to the specific coupling of repair to arrested transcription by RNA polymerase. The biochemical basis for strand-specific repair in eukaryotes is unknown. Second, in eukaryotes some or all of the subunits of transcription factor IIH (TFIIH) are required for nucleotide excision repair. The biological significance of this dual function of TFIIH proteins is not obvious. Finally, there are indications that the genes CSA and CSB, which are implicated in the human hereditary disease Cockayne syndrome, may have a role in transcription.

DNA Damage↗
PubMed1995

Role of the Rad1 and Rad10 proteins in nucleotide excision repair and recombination.

In Saccharomyces cerevisiae, the RAD1 and RAD10 genes are involved in DNA nucleotide excision repair (NER) and in a pathway of mitotic recombination that occurs between direct repeat DNA sequences. In this paper, we show that purified Rad1 and Rad10 interact with a synthetic bubble structure and incise the DNA at the 5'-side of the centrally unpaired region. When Rad1-Rad10 and purified XPG protein (the human homolog of yeast Rad2 protein) were co-incubated with the DNA substrate, we observed incisions at both ends of the bubble. This reaction mimics the dual incision step in nucleotide excision repair in vivo. In addition, the recent suggestion that Rad1 can act to resolve Holliday junctions (Habraken, Y., Sung, P., Prakash, L., and Prakash, S. (1994) Nature 371, 531-534), explaining the recombination defect observed in rad1 mutants, has been further investigated. However, using proteins purified in two different laboratories we were unable to show any interaction between Rad1 and synthetic Holliday junctions. The role that Rad1-Rad10 plays in recombination is likely to resemble its activity in NER by acting upon partially unpaired DNA intermediates such as those formed by recombination mechanisms involving single-strand DNA annealing.

DNA Repair↗