P. Sung - Academia.edu (original) (raw)

Papers by P. Sung

Research paper thumbnail of Specific complex formation between proteins encoded by the yeast DNA repair and recombination genes RAD1 and RAD10

Proceedings of the National Academy of Sciences, 1992

The RADI and RADIO genes of Saccharomyces cerevisiae are required for excision repair of ultravio... more The RADI and RADIO genes of Saccharomyces cerevisiae are required for excision repair of ultraviolet light-damaged DNA, and they also function in a mitotic recombination pathway that is distinct from the double-strandbreak recombination pathway controlled by RAD52. Here, we show that the RAD1 and RADi1 proteins are complexed with each other in vivo. Immunoprecipitation of yeast cell extracts with either anti-RAD1 antibody or anti-RAD10 antibody coprecipitated quantitative amounts of both RAD1 and RADiO proteins. The level of coprecipitable RAD1 and RADiO increased when both proteins were overproduced together, but not if only one of the proteins was overproduced. The RAD1/RAD10 complex is highly stable, being refractory to 1 M NaCl and to low concentrations of SDS. By hydroxylamine mutagenesis, we have identified a radi mutant allele whose encoded protein fails to complex with RADiO. The interactiondefective radi mutant resembles the radi or radiO null mutant in defective DNA repair and recombination, implying that complex formation is essential for the expression of biological activities controlled by RADI and RADIO.

Research paper thumbnail of Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision repair factor 3: implications for nucleotide excision repair and Cockayne syndrome

Proceedings of the National Academy of Sciences, 1996

Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large ... more Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large number of highly conserved protein factors. Recent studies in yeast have suggested that NER involves the action of distinct protein subassemblies at the damage site rather than the placement there of a "preformed repairosome" containing all the essential NER factors. Neither of the two endonucleases, Rad1-Rad10 and Rad2, required for dual incision, shows any affinity for ultraviolet-damaged DNA. Rad1-Rad10 forms a ternary complex with the DNA damage recognition protein Rad14, providing a means for targeting this nuclease to the damage site. It has remained unclear how the Rad2 nuclease is targeted to the DNA damage site and why mutations in the human RAD2 counterpart, XPG, result in Cockayne syndrome. Here we examine whether Rad2 is part of a higher order subassembly. Interestingly, we find copurification of Rad2 protein with TFIIH, such that TFIIH purified from a strain that overexpresses Rad2 contains a stoichiometric amount of Rad2. By several independent criteria, we establish that Rad2 is tightly associated with TFIIH, exhibiting an apparent dissociation constant < 3.3 x 10(-9) M. These results identify a novel subassembly consisting of TFIIH and Rad2, which we have designated as nucleotide excision repair factor 3. Association with TFIIH provides a means of targeting Rad2 to the damage site, where its endonuclease activity would mediate the 3' incision. Our findings are important for understanding the manner of assembly of the NER machinery and they have implications for Cockayne syndrome.

Research paper thumbnail of Rad54p Is a Chromatin Remodeling Enzyme Required for Heteroduplex DNA Joint Formation with Chromatin

Journal of Biological Chemistry, 2003

In eukaryotic cells, the repair of DNA double-strand breaks by homologous recombination requires ... more In eukaryotic cells, the repair of DNA double-strand breaks by homologous recombination requires a RecAlike recombinase, Rad51p, and a Swi2p/Snf2p-like ATPase, Rad54p. Here we find that yeast Rad51p and Rad54p support robust homologous pairing between single-stranded DNA and a chromatin donor. In contrast, bacterial RecA is incapable of catalyzing homologous pairing with a chromatin donor. We also show that Rad54p possesses many of the biochemical properties of bona fide ATP-dependent chromatin-remodeling enzymes, such as ySWI/SNF. Rad54p can enhance the accessibility of DNA within nucleosomal arrays, but it does not seem to disrupt nucleosome positioning. Taken together, our results indicate that Rad54p is a chromatinremodeling enzyme that promotes homologous DNA pairing events within the context of chromatin.

Research paper thumbnail of Trisomy 21 mid-trimester amniotic fluid induced pluripotent stem cells maintain genetic signatures during reprogramming: implications for disease modeling and cryobanking

Trisomy 21 is the most common chromosomal abnormality and is associated primarily with cardiovasc... more Trisomy 21 is the most common chromosomal abnormality and is associated primarily with cardiovascular, hematological, and neurological complications. A robust patient-derived cellular model is necessary to investigate the pathophysiology of the syndrome because current animal models are limited and access to tissues from affected individuals is ethically challenging. We aimed to derive induced pluripotent stem cells (iPSCs) from trisomy 21 human mid-trimester amniotic fluid stem cells (AFSCs) and describe their hematopoietic and neurological characteristics. Human AFSCs collected from women undergoing prenatal diagnosis were selected for c-KIT(+) and transduced with a Cre-lox-inducible polycistronic lentiviral vector encoding SOX2, OCT4, KLF-4, and c-MYC (50,000 cells at a multiplicity of infection (MOI) 1-5 for 72 h). The embryonic stem cell (ESC)-like properties of the AFSC-derived iPSCs were established in vitro by embryoid body formation and in vivo by teratoma formation in RAG2(-/-), γ-chain(-/-), C2(-/-) immunodeficient mice. Reprogrammed cells retained their cytogenetic signatures and differentiated into specialized hematopoietic and neural precursors detected by morphological assessment, immunostaining, and RT-PCR. Additionally, the iPSCs expressed all pluripotency markers upon multiple rounds of freeze-thawing. These findings are important in establishing a patient-specific cellular platform of trisomy 21 to study the pathophysiology of the aneuploidy and for future drug discovery.

Research paper thumbnail of Specific complex formation between proteins encoded by the yeast DNA repair and recombination genes RAD1 and RAD10

Proceedings of the National Academy of Sciences, 1992

The RADI and RADIO genes of Saccharomyces cerevisiae are required for excision repair of ultravio... more The RADI and RADIO genes of Saccharomyces cerevisiae are required for excision repair of ultraviolet light-damaged DNA, and they also function in a mitotic recombination pathway that is distinct from the double-strandbreak recombination pathway controlled by RAD52. Here, we show that the RAD1 and RADi1 proteins are complexed with each other in vivo. Immunoprecipitation of yeast cell extracts with either anti-RAD1 antibody or anti-RAD10 antibody coprecipitated quantitative amounts of both RAD1 and RADiO proteins. The level of coprecipitable RAD1 and RADiO increased when both proteins were overproduced together, but not if only one of the proteins was overproduced. The RAD1/RAD10 complex is highly stable, being refractory to 1 M NaCl and to low concentrations of SDS. By hydroxylamine mutagenesis, we have identified a radi mutant allele whose encoded protein fails to complex with RADiO. The interactiondefective radi mutant resembles the radi or radiO null mutant in defective DNA repair and recombination, implying that complex formation is essential for the expression of biological activities controlled by RADI and RADIO.

Research paper thumbnail of Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision repair factor 3: implications for nucleotide excision repair and Cockayne syndrome

Proceedings of the National Academy of Sciences, 1996

Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large ... more Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large number of highly conserved protein factors. Recent studies in yeast have suggested that NER involves the action of distinct protein subassemblies at the damage site rather than the placement there of a "preformed repairosome" containing all the essential NER factors. Neither of the two endonucleases, Rad1-Rad10 and Rad2, required for dual incision, shows any affinity for ultraviolet-damaged DNA. Rad1-Rad10 forms a ternary complex with the DNA damage recognition protein Rad14, providing a means for targeting this nuclease to the damage site. It has remained unclear how the Rad2 nuclease is targeted to the DNA damage site and why mutations in the human RAD2 counterpart, XPG, result in Cockayne syndrome. Here we examine whether Rad2 is part of a higher order subassembly. Interestingly, we find copurification of Rad2 protein with TFIIH, such that TFIIH purified from a strain that overexpresses Rad2 contains a stoichiometric amount of Rad2. By several independent criteria, we establish that Rad2 is tightly associated with TFIIH, exhibiting an apparent dissociation constant < 3.3 x 10(-9) M. These results identify a novel subassembly consisting of TFIIH and Rad2, which we have designated as nucleotide excision repair factor 3. Association with TFIIH provides a means of targeting Rad2 to the damage site, where its endonuclease activity would mediate the 3' incision. Our findings are important for understanding the manner of assembly of the NER machinery and they have implications for Cockayne syndrome.

Research paper thumbnail of Rad54p Is a Chromatin Remodeling Enzyme Required for Heteroduplex DNA Joint Formation with Chromatin

Journal of Biological Chemistry, 2003

In eukaryotic cells, the repair of DNA double-strand breaks by homologous recombination requires ... more In eukaryotic cells, the repair of DNA double-strand breaks by homologous recombination requires a RecAlike recombinase, Rad51p, and a Swi2p/Snf2p-like ATPase, Rad54p. Here we find that yeast Rad51p and Rad54p support robust homologous pairing between single-stranded DNA and a chromatin donor. In contrast, bacterial RecA is incapable of catalyzing homologous pairing with a chromatin donor. We also show that Rad54p possesses many of the biochemical properties of bona fide ATP-dependent chromatin-remodeling enzymes, such as ySWI/SNF. Rad54p can enhance the accessibility of DNA within nucleosomal arrays, but it does not seem to disrupt nucleosome positioning. Taken together, our results indicate that Rad54p is a chromatinremodeling enzyme that promotes homologous DNA pairing events within the context of chromatin.

Research paper thumbnail of Trisomy 21 mid-trimester amniotic fluid induced pluripotent stem cells maintain genetic signatures during reprogramming: implications for disease modeling and cryobanking

Trisomy 21 is the most common chromosomal abnormality and is associated primarily with cardiovasc... more Trisomy 21 is the most common chromosomal abnormality and is associated primarily with cardiovascular, hematological, and neurological complications. A robust patient-derived cellular model is necessary to investigate the pathophysiology of the syndrome because current animal models are limited and access to tissues from affected individuals is ethically challenging. We aimed to derive induced pluripotent stem cells (iPSCs) from trisomy 21 human mid-trimester amniotic fluid stem cells (AFSCs) and describe their hematopoietic and neurological characteristics. Human AFSCs collected from women undergoing prenatal diagnosis were selected for c-KIT(+) and transduced with a Cre-lox-inducible polycistronic lentiviral vector encoding SOX2, OCT4, KLF-4, and c-MYC (50,000 cells at a multiplicity of infection (MOI) 1-5 for 72 h). The embryonic stem cell (ESC)-like properties of the AFSC-derived iPSCs were established in vitro by embryoid body formation and in vivo by teratoma formation in RAG2(-/-), γ-chain(-/-), C2(-/-) immunodeficient mice. Reprogrammed cells retained their cytogenetic signatures and differentiated into specialized hematopoietic and neural precursors detected by morphological assessment, immunostaining, and RT-PCR. Additionally, the iPSCs expressed all pluripotency markers upon multiple rounds of freeze-thawing. These findings are important in establishing a patient-specific cellular platform of trisomy 21 to study the pathophysiology of the aneuploidy and for future drug discovery.