The transcriptional coactivator p300 interacts with many transcription factors that participate i... more The transcriptional coactivator p300 interacts with many transcription factors that participate in a broad spectrum of biological activities, such as cellular differentiation, homeostasis and growth control. Mouse embryos lacking both p300 alleles die around mid-gestation, with pleiotropic defects in morphogenesis, in cell differentiation and, unexpectedly, in cell proliferation because of reduced DNA synthesis. Here we show that p300 may have a role in DNA repair synthesis through its interaction with proliferating cell nuclear antigen (PCNA). We show that in vitro and in vivo p300 forms a complex with PCNA that does not depend on the S phase of cell cycle. A large fraction of both p300 and PCNA colocalize to speckled structures in the nucleus. Furthermore, the endogenous p300-PCNA complex stimulates DNA synthesis in vitro. Chromatin immunoprecipitation experiments indicate that p300 is associated with freshly synthesized DNA after ultraviolet irradiation. Our results suggest that ...
The International Journal of Biochemistry & Cell Biology, 2008
Poly-ADP-ribose polymerase-2 (PARP-2) was described to regulate cellular functions comprising DNA... more Poly-ADP-ribose polymerase-2 (PARP-2) was described to regulate cellular functions comprising DNA surveillance, inflammation and cell differentiation by co-regulating different transcription factors. Using an in vitro and in vivo approach, we identified PARP-2 as a new substrate for the histone acetyltransferases PCAF and GCN5L. Site directed mutagenesis indicated that lysines 36 and 37, located in the nuclear localization signal of PARP-2, are the main targets for PCAF and GCN5L activity in vitro. Interestingly, acetylation of the same two PARP-2 residues reduces the DNA binding and enzymatic activity of PARP-2. Finally, PARP-2 with mutated lysines 36 and 37 showed reduced auto-mono-ADP-ribosylation when compared to wild type PARP-2. Together, our results provide evidence that acetylation of PARP-2 is a key post-translational modification that may regulate DNA binding and consequently also the enzymatic activity of PARP-2.
DNA polymerase beta (pol beta) is a key player in DNA base excision repair (BER). Here, we descri... more DNA polymerase beta (pol beta) is a key player in DNA base excision repair (BER). Here, we describe the complex formation of pol beta with the protein arginine methyltransferase 1 (PRMT1). PRMT1 specifically methylated pol beta in vitro and in vivo. Arginine 137 was identified in pol beta as an important target for methylation by PRMT1. Neither the polymerase nor the dRP-lyase activities of pol beta were affected by PRMT1 methylation. However, this modification abolished the interaction of pol beta with proliferating cell nuclear antigen (PCNA). Together, our results provide evidence that PRMT1 methylation of pol beta might play a regulatory role in BER by preventing the involvement of pol beta in PCNA-dependent DNA metabolic events.
Polyphosphate (polyP) occurs ubiquitously in cells, but its functions are poorly understood and i... more Polyphosphate (polyP) occurs ubiquitously in cells, but its functions are poorly understood and its synthesis has only been characterized in bacteria. Using x-ray crystallography, we identified a eukaryotic polyphosphate polymerase within the membrane-integral vacuolar transporter chaperone (VTC) complex. A 2.6 angstrom crystal structure of the catalytic domain grown in the presence of adenosine triphosphate (ATP) reveals polyP winding through a tunnel-shaped pocket. Nucleotide- and phosphate-bound structures suggest that the enzyme functions by metal-assisted cleavage of the ATP gamma-phosphate, which is then in-line transferred to an acceptor phosphate to form polyP chains. Mutational analysis of the transmembrane domain indicates that VTC may integrate cytoplasmic polymer synthesis with polyP membrane translocation. Identification of the polyP-synthesizing enzyme opens the way to determine the functions of polyP in lower eukaryotes.
Alterations in DNA repair lead to genomic instability and higher risk of cancer. DNA base excisio... more Alterations in DNA repair lead to genomic instability and higher risk of cancer. DNA base excision repair (BER) corrects damaged bases, apurinic sites, and single-strand DNA breaks. Here, a regulatory mechanism for DNA polymerase beta (Pol beta) is described. Pol beta was found to form a complex with the protein arginine methyltransferase 6 (PRMT6) and was specifically methylated in vitro and in vivo. Methylation of Pol beta by PRMT6 strongly stimulated DNA polymerase activity by enhancing DNA binding and processivity, while single nucleotide insertion and dRP-lyase activity were not affected. Two residues, R83 and R152, were identified in Pol beta as the sites of methylation by PRMT6. Genetic complementation of Pol beta knockout cells with R83/152K mutant revealed the importance of these residues for the cellular resistance to DNA alkylating agent. Based on our findings, we propose that PRMT6 plays a role as a regulator of BER.
We describe a role for the transcriptional coactivator p300 in DNA metabolism. p300 formed a comp... more We describe a role for the transcriptional coactivator p300 in DNA metabolism. p300 formed a complex with flap endonuclease-1 (Fen1) and acetylated Fen1 in vitro. Furthermore, Fen1 acetylation was observed in vivo and was enhanced upon UV treatment of human cells. Remarkably, acetylation of the Fen1 C terminus by p300 significantly reduced Fen1's DNA binding and nuclease activity. Proliferating cell nuclear antigen (PCNA) was able to stimulate both acetylated and unacetylated Fen1 activity to the same extent. Our results identify acetylation as a novel regulatory modification of Fen1 and implicate that p300 is not only a component of the chromatin remodeling machinery but might also play a critical role in regulating DNA metabolic events.
Hypoxia-inducible factor 1 (HIF-1) is the key transcription factor regulating hypoxia-dependent g... more Hypoxia-inducible factor 1 (HIF-1) is the key transcription factor regulating hypoxia-dependent gene expression. Lack of oxygen stabilizes HIF-1, which in turn modulates the gene expression pattern to adapt cells to the hypoxic environment. Activation of HIF-1 is also detected in most solid tumors and supports tumor growth through the expression of target genes that are involved in processes like cell proliferation, energy metabolism, and oxygen delivery. Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated protein, which was shown to regulate transcription. Here we report that chronic myelogenous leukemia cells expressing small interfering RNA against PARP1, which were injected into wild-type mice expressing PARP1, showed tumor growth with increased levels of necrosis, limited vascularization, and reduced expression of GLUT-1. Of note, PARP1-deficient cells showed a reduced HIF-1 transcriptional activation that was dependent on PARP1 enzymatic activity. PARP1 neither influenced binding of HIF-1 to its hypoxic response element nor changed HIF-1alpha protein levels in hypoxic cells. However, PARP1 formed a complex with HIF-1alpha through direct protein interaction and coactivated HIF-1alpha-dependent gene expression. These findings provide convincing evidence that wild-type mice expressing PARP1 cannot compensate for the loss of PARP1 in tumor cells and strengthen the importance of the role of PARP1 as a transcriptional coactivator of HIF-1-dependent gene expression during tumor progression.
The transcriptional coactivator p300 interacts with many transcription factors that participate i... more The transcriptional coactivator p300 interacts with many transcription factors that participate in a broad spectrum of biological activities, such as cellular differentiation, homeostasis and growth control. Mouse embryos lacking both p300 alleles die around mid-gestation, with pleiotropic defects in morphogenesis, in cell differentiation and, unexpectedly, in cell proliferation because of reduced DNA synthesis. Here we show that p300 may have a role in DNA repair synthesis through its interaction with proliferating cell nuclear antigen (PCNA). We show that in vitro and in vivo p300 forms a complex with PCNA that does not depend on the S phase of cell cycle. A large fraction of both p300 and PCNA colocalize to speckled structures in the nucleus. Furthermore, the endogenous p300-PCNA complex stimulates DNA synthesis in vitro. Chromatin immunoprecipitation experiments indicate that p300 is associated with freshly synthesized DNA after ultraviolet irradiation. Our results suggest that ...
The International Journal of Biochemistry & Cell Biology, 2008
Poly-ADP-ribose polymerase-2 (PARP-2) was described to regulate cellular functions comprising DNA... more Poly-ADP-ribose polymerase-2 (PARP-2) was described to regulate cellular functions comprising DNA surveillance, inflammation and cell differentiation by co-regulating different transcription factors. Using an in vitro and in vivo approach, we identified PARP-2 as a new substrate for the histone acetyltransferases PCAF and GCN5L. Site directed mutagenesis indicated that lysines 36 and 37, located in the nuclear localization signal of PARP-2, are the main targets for PCAF and GCN5L activity in vitro. Interestingly, acetylation of the same two PARP-2 residues reduces the DNA binding and enzymatic activity of PARP-2. Finally, PARP-2 with mutated lysines 36 and 37 showed reduced auto-mono-ADP-ribosylation when compared to wild type PARP-2. Together, our results provide evidence that acetylation of PARP-2 is a key post-translational modification that may regulate DNA binding and consequently also the enzymatic activity of PARP-2.
DNA polymerase beta (pol beta) is a key player in DNA base excision repair (BER). Here, we descri... more DNA polymerase beta (pol beta) is a key player in DNA base excision repair (BER). Here, we describe the complex formation of pol beta with the protein arginine methyltransferase 1 (PRMT1). PRMT1 specifically methylated pol beta in vitro and in vivo. Arginine 137 was identified in pol beta as an important target for methylation by PRMT1. Neither the polymerase nor the dRP-lyase activities of pol beta were affected by PRMT1 methylation. However, this modification abolished the interaction of pol beta with proliferating cell nuclear antigen (PCNA). Together, our results provide evidence that PRMT1 methylation of pol beta might play a regulatory role in BER by preventing the involvement of pol beta in PCNA-dependent DNA metabolic events.
Polyphosphate (polyP) occurs ubiquitously in cells, but its functions are poorly understood and i... more Polyphosphate (polyP) occurs ubiquitously in cells, but its functions are poorly understood and its synthesis has only been characterized in bacteria. Using x-ray crystallography, we identified a eukaryotic polyphosphate polymerase within the membrane-integral vacuolar transporter chaperone (VTC) complex. A 2.6 angstrom crystal structure of the catalytic domain grown in the presence of adenosine triphosphate (ATP) reveals polyP winding through a tunnel-shaped pocket. Nucleotide- and phosphate-bound structures suggest that the enzyme functions by metal-assisted cleavage of the ATP gamma-phosphate, which is then in-line transferred to an acceptor phosphate to form polyP chains. Mutational analysis of the transmembrane domain indicates that VTC may integrate cytoplasmic polymer synthesis with polyP membrane translocation. Identification of the polyP-synthesizing enzyme opens the way to determine the functions of polyP in lower eukaryotes.
Alterations in DNA repair lead to genomic instability and higher risk of cancer. DNA base excisio... more Alterations in DNA repair lead to genomic instability and higher risk of cancer. DNA base excision repair (BER) corrects damaged bases, apurinic sites, and single-strand DNA breaks. Here, a regulatory mechanism for DNA polymerase beta (Pol beta) is described. Pol beta was found to form a complex with the protein arginine methyltransferase 6 (PRMT6) and was specifically methylated in vitro and in vivo. Methylation of Pol beta by PRMT6 strongly stimulated DNA polymerase activity by enhancing DNA binding and processivity, while single nucleotide insertion and dRP-lyase activity were not affected. Two residues, R83 and R152, were identified in Pol beta as the sites of methylation by PRMT6. Genetic complementation of Pol beta knockout cells with R83/152K mutant revealed the importance of these residues for the cellular resistance to DNA alkylating agent. Based on our findings, we propose that PRMT6 plays a role as a regulator of BER.
We describe a role for the transcriptional coactivator p300 in DNA metabolism. p300 formed a comp... more We describe a role for the transcriptional coactivator p300 in DNA metabolism. p300 formed a complex with flap endonuclease-1 (Fen1) and acetylated Fen1 in vitro. Furthermore, Fen1 acetylation was observed in vivo and was enhanced upon UV treatment of human cells. Remarkably, acetylation of the Fen1 C terminus by p300 significantly reduced Fen1's DNA binding and nuclease activity. Proliferating cell nuclear antigen (PCNA) was able to stimulate both acetylated and unacetylated Fen1 activity to the same extent. Our results identify acetylation as a novel regulatory modification of Fen1 and implicate that p300 is not only a component of the chromatin remodeling machinery but might also play a critical role in regulating DNA metabolic events.
Hypoxia-inducible factor 1 (HIF-1) is the key transcription factor regulating hypoxia-dependent g... more Hypoxia-inducible factor 1 (HIF-1) is the key transcription factor regulating hypoxia-dependent gene expression. Lack of oxygen stabilizes HIF-1, which in turn modulates the gene expression pattern to adapt cells to the hypoxic environment. Activation of HIF-1 is also detected in most solid tumors and supports tumor growth through the expression of target genes that are involved in processes like cell proliferation, energy metabolism, and oxygen delivery. Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated protein, which was shown to regulate transcription. Here we report that chronic myelogenous leukemia cells expressing small interfering RNA against PARP1, which were injected into wild-type mice expressing PARP1, showed tumor growth with increased levels of necrosis, limited vascularization, and reduced expression of GLUT-1. Of note, PARP1-deficient cells showed a reduced HIF-1 transcriptional activation that was dependent on PARP1 enzymatic activity. PARP1 neither influenced binding of HIF-1 to its hypoxic response element nor changed HIF-1alpha protein levels in hypoxic cells. However, PARP1 formed a complex with HIF-1alpha through direct protein interaction and coactivated HIF-1alpha-dependent gene expression. These findings provide convincing evidence that wild-type mice expressing PARP1 cannot compensate for the loss of PARP1 in tumor cells and strengthen the importance of the role of PARP1 as a transcriptional coactivator of HIF-1-dependent gene expression during tumor progression.
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