Vincenzo Salerno - Academia.edu (original) (raw)

Papers by Vincenzo Salerno

Research paper thumbnail of The mismatch repair-mediated cell cycle checkpoint response to fluorodeoxyuridine

Journal of Cellular Biochemistry, Jun 9, 2008

The loss of DNA mismatch repair (MMR) is responsible for hereditary nonpolyposis colorectal cance... more The loss of DNA mismatch repair (MMR) is responsible for hereditary nonpolyposis colorectal cancer and a subset of sporadic tumors. Acquired resistance or tolerance to some anti-cancer drugs occurs when MMR function is impaired. 5-Fluorouracil (FU), an anti-cancer drug used in the treatment of advanced colorectal and other cancers, and its metabolites are incorporated into RNA and DNA and inhibit thymidylate synthase resulting in depletion of dTTP and incorporation in DNA of uracil. Although the MMR deficiency has been implicated in tolerance to FU, the mechanism of cell killing remains unclear. Here, we examine the cellular response to fluorodeoxyuridine (FdU) and the role of the MMR system. After brief exposure of cells to low doses of FdU, MMR mediates DNA damage signaling during S-phase and triggers arrest in G2/M in the first cell cycle in a manner requiring MutSα, MutLα, and DNA replication. Cell cycle arrest is mediated by ATR kinase and results in phosphorylation of Chk1 and SMC1. MutSα binds FdU:G mispairs in vitro consistent with its being a DNA damage sensor. Prolonged treatment with FdU results in an irreversible arrest in G2 that is independent of MMR status and leads to the accumulation of DNA lesions that are targeted by the base excision repair (BER) pathway. Thus, MMR can act as a direct sensor of FdU-mediated DNA lesions eliciting cell cycle arrest via the ATR/Chk1 pathway. However, at higher levels of damage, other damage surveillance pathways such as BER also play important roles.

Research paper thumbnail of In vitro studies of DNA mismatch repair proteins

Analytical Biochemistry, Jun 1, 2011

The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells i... more The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells is important for understanding mechanisms involved in mutagenesis and tumorigenesis. Since mismatch repair proteins recognize mismatches containing both normal and chemically altered or damaged bases, in vitro assays must accommodate a variety of mismatches in different sequence contexts. Here we describe the construction of DNA mismatch substrates containing G:T or O 6 meG:T mismatches, the purification of recombinant native human MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) proteins, and in vitro mismatch repair and excision assays that can be adapted to study mismatch repair in nuclear extracts from mismatch repair proficient and deficient cells.

Research paper thumbnail of In vitro studies of DNA mismatch repair proteins

Analytical Biochemistry, 2011

The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells i... more The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells is important for understanding mechanisms involved in mutagenesis and tumorigenesis. Since mismatch repair proteins recognize mismatches containing both normal and chemically altered or damaged bases, in vitro assays must accommodate a variety of mismatches in different sequence contexts. Here we describe the construction of DNA mismatch substrates containing G:T or O 6 meG:T mismatches, the purification of recombinant native human MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) proteins, and in vitro mismatch repair and excision assays that can be adapted to study mismatch repair in nuclear extracts from mismatch repair proficient and deficient cells.

Research paper thumbnail of Lack of Strand-specific Repair of UV-induced DNA Lesions in Three Genes of the Archaeon Sulfolobus solfataricus

Journal of Molecular Biology, 2007

In all organisms, specialized systems are devoted to repair of DNA lesions induced by exposure to... more In all organisms, specialized systems are devoted to repair of DNA lesions induced by exposure to UV light. In both Eucarya and Bacteria, UV-induced pyrimidine dimers in the transcribed strand of active genes are repaired at a faster rate compared to the non-transcribed strand and the rest of the genome. Preferential repair of transcribed strands requires the Transcription-Repair Coupling Factor in Escherichia coli and the CSA and CSB proteins in humans. These factors are needed for coupling of transcription to nucleotide excision repair (NER), a major pathway for repair of UVinduced lesions. Whereas transcription-coupled NER (TC-NER) is an evolutionary conserved process, not all active genes show preferential repair of transcribed strands. The existence of a NER pathway in the Archaea has not been demonstrated directly, yet it is suggested by the presence and properties of homologues of NER nucleases and helicases. However, none of the proteins responsible for the lesion recognition steps or for TC-NER has been found in archaeal genomes. Moreover, the kinetics of gene or strand-specific repair has never been investigated in any organism of this domain. We have analysed the kinetics of repair of UV-induced DNA damage in the transcribed and non-transcribed strands of three genes of the hyperthermophilic archaeon Sulfolobus solfataricus. We found that in all three genes the two strands are repaired with the same efficiency with each other and with the genome in general, thus providing no evidence of strand bias or transcription coupling of the repair process in the genes analysed. Further studies will be required to test the existence of a transcriptioncoupled repair pathway in other archaeal genes and to elucidate the mechanism of UV lesion recognition and repair in Archaea.

Research paper thumbnail of Lack of strand-specific repair of UV-induced DNA lesions in three genes of the archaeon Sulfolobus solfataricus

Journal of molecular biology, Jan 26, 2007

In all organisms, specialized systems are devoted to repair of DNA lesions induced by exposure to... more In all organisms, specialized systems are devoted to repair of DNA lesions induced by exposure to UV light. In both Eucarya and Bacteria, UV-induced pyrimidine dimers in the transcribed strand of active genes are repaired at a faster rate compared to the non-transcribed strand and the rest of the genome. Preferential repair of transcribed strands requires the Transcription-Repair Coupling Factor in Escherichia coli and the CSA and CSB proteins in humans. These factors are needed for coupling of transcription to nucleotide excision repair (NER), a major pathway for repair of UV-induced lesions. Whereas transcription-coupled NER (TC-NER) is an evolutionary conserved process, not all active genes show preferential repair of transcribed strands. The existence of a NER pathway in the Archaea has not been demonstrated directly, yet it is suggested by the presence and properties of homologues of NER nucleases and helicases. However, none of the proteins responsible for the lesion recogniti...

Research paper thumbnail of Cytokine Immunotherapy

Experimental and Applied Immunotherapy, 2010

... M. Nelles (*) Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada e-... more ... M. Nelles (*) Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada e-mail: mnelles@uhnres.utoronto.ca Chapter 13 Cytokine Immunotherapy Megan Nelles, Vincenzo Salerno, Yixin Xu, and Christopher J. Paige Page 2. 282 M. Nelles et al. Introduction ...

Research paper thumbnail of Functional interaction of reverse gyrase with single-strand binding protein of the archaeon Sulfolobus

Nucleic Acids Research, 2005

Reverse gyrase is a unique hyperthermophilespecific DNA topoisomerase that induces positive super... more Reverse gyrase is a unique hyperthermophilespecific DNA topoisomerase that induces positive supercoiling. It is a modular enzyme composed of a topoisomerase IA and a helicase domain, which cooperate in the ATP-dependent positive supercoiling reaction. Although its physiological function has not been determined, it can be hypothesized that, like the topoisomerase-helicase complexes found in every organism, reverse gyrase might participate in different DNA transactions mediated by multiprotein complexes. Here, we show that reverse gyrase activity is stimulated by the single-strand binding protein (SSB) from the archaeon Sulfolobus solfataricus. Using a combination of in vitro assays we analysed each step of the complex reverse gyrase reaction. SSB stimulates all the steps of the reaction: binding to DNA, DNA cleavage, strand passage and ligation. By co-immunoprecipitation of cell extracts we show that reverse gyrase and SSB assemble a complex in the presence of DNA, but do not make stable protein-protein interactions. In addition, SSB stimulates reverse gyrase positive supercoiling activity on DNA templates associated with the chromatin protein Sul7d. Furthermore, SSB enhances binding and cleavage of UV-irradiated substrates by reverse gyrase. The results shown here suggest that these functional interactions may have biological relevance and that the interplay of different DNA binding proteins might modulate reverse gyrase activity in DNA metabolic pathways.

Research paper thumbnail of Transcriptional response to DNA damage in the archaeon Sulfolobus solfataricus

Nucleic Acids Research, 2003

Exposure of cells to DNA-damaging agents triggers a complex biological response involving cell cy... more Exposure of cells to DNA-damaging agents triggers a complex biological response involving cell cycle arrest and modulation of gene expression. Genomic sequencing has revealed the presence of archaeal genes homologous to components of the eucaryal nucleotide excision repair (NER) pathway, which is involved in the repair of ultraviolet (UV) lightinduced DNA damage. However, the events involved in the cell response to UV irradiation and their regulation have not been studied in Archaea. We show here that UV radiation induces the formation of cyclobutane pyrimidine dimers (CPDs) in the hyperthermophilic archaeon Sulfolobus solfataricus, and that these lesions are ef®ciently repaired in vivo in the dark, suggesting that a NER pathway is active. DNA damage is a signal for concomitant growth arrest and transcriptional induction of the NER genes XPF, XPG and XPB. The cell response to UV irradiation includes transcriptional regulation of genes encoding two DNA binding proteins involved in chromosome dynamics. Moreover, several of these genes are also strongly induced by the intercalating agent actinomycin D. Thus, response to DNA damage in S.solfataricus has features essentially conserved in all three domains of life.

Research paper thumbnail of Lack of Strand-specific Repair of UV-induced DNA Lesions in Three Genes of the Archaeon Sulfolobus solfataricus

Journal of Molecular Biology, 2007

In all organisms, specialized systems are devoted to repair of DNA lesions induced by exposure to... more In all organisms, specialized systems are devoted to repair of DNA lesions induced by exposure to UV light. In both Eucarya and Bacteria, UV-induced pyrimidine dimers in the transcribed strand of active genes are repaired at a faster rate compared to the non-transcribed strand and the rest of the genome. Preferential repair of transcribed strands requires the Transcription-Repair Coupling Factor in Escherichia coli and the CSA and CSB proteins in humans. These factors are needed for coupling of transcription to nucleotide excision repair (NER), a major pathway for repair of UV-induced lesions. Whereas transcription-coupled NER (TC-NER) is an evolutionary conserved process, not all active genes show preferential repair of transcribed strands. The existence of a NER pathway in the Archaea has not been demonstrated directly, yet it is suggested by the presence and properties of homologues of NER nucleases and helicases. However, none of the proteins responsible for the lesion recognition steps or for TC-NER has been found in archaeal genomes. Moreover, the kinetics of gene or strand-specific repair has never been investigated in any organism of this domain. We have analysed the kinetics of repair of UV-induced DNA damage in the transcribed and non-transcribed strands of three genes of the hyperthermophilic archaeon Sulfolobus solfataricus. We found that in all three genes the two strands are repaired with the same efficiency with each other and with the genome in general, thus providing no evidence of strand bias or transcription coupling of the repair process in the genes analysed. Further studies will be required to test the existence of a transcription-coupled repair pathway in other archaeal genes and to elucidate the mechanism of UV lesion recognition and repair in Archaea.

Research paper thumbnail of The mismatch repair-mediated cell cycle checkpoint response to fluorodeoxyuridine

Journal of Cellular Biochemistry, 2008

The loss of DNA mismatch repair (MMR) is responsible for hereditary nonpolyposis colorectal cance... more The loss of DNA mismatch repair (MMR) is responsible for hereditary nonpolyposis colorectal cancer and a subset of sporadic tumors. Acquired resistance or tolerance to some anti-cancer drugs occurs when MMR function is impaired. 5-Fluorouracil (FU), an anti-cancer drug used in the treatment of advanced colorectal and other cancers, and its metabolites are incorporated into RNA and DNA and inhibit thymidylate synthase resulting in depletion of dTTP and incorporation in DNA of uracil. Although the MMR deficiency has been implicated in tolerance to FU, the mechanism of cell killing remains unclear. Here, we examine the cellular response to fluorodeoxyuridine (FdU) and the role of the MMR system. After brief exposure of cells to low doses of FdU, MMR mediates DNA damage signaling during S-phase and triggers arrest in G2/M in the first cell cycle in a manner requiring MutSα, MutLα, and DNA replication. Cell cycle arrest is mediated by ATR kinase and results in phosphorylation of Chk1 and SMC1. MutSα binds FdU:G mispairs in vitro consistent with its being a DNA damage sensor. Prolonged treatment with FdU results in an irreversible arrest in G2 that is independent of MMR status and leads to the accumulation of DNA lesions that are targeted by the base excision repair (BER) pathway. Thus, MMR can act as a direct sensor of FdU-mediated DNA lesions eliciting cell cycle arrest via the ATR/Chk1 pathway. However, at higher levels of damage, other damage surveillance pathways such as BER also play important roles.

Research paper thumbnail of Reverse Gyrase Recruitment to DNA after UV Light Irradiation in Sulfolobus solfataricus

Journal of Biological Chemistry, 2004

Induction of DNA damage triggers a complex biological response concerning not only repair systems... more Induction of DNA damage triggers a complex biological response concerning not only repair systems but also virtually every cell function. DNA topoisomerases regulate the level of DNA supercoiling in all DNA transactions. Reverse gyrase is a peculiar DNA topoisomerase, specific to hyperthermophilic microorganisms, which contains a helicase and a topoisomerase IA domain that has the unique ability to introduce positive supercoiling into DNA molecules. We show here that reverse gyrase of the archaean Sulfolobus solfataricus is mobilized to DNA in vivo after UV irradiation. The enzyme, either purified or in cell extracts, forms stable covalent complexes with UV-damaged DNA in vitro. We also show that the reverse gyrase translocation to DNA in vivo and the stabilization of covalent complexes in vitro are specific effects of UV light irradiation and do not occur with the intercalating agent actinomycin D. Our results suggest that reverse gyrase might participate, directly or indirectly, in the cell response to UV light-induced DNA damage. This is the first direct evidence of the recruitment of a topoisomerase IA enzyme to DNA after the induction of DNA damage. The interaction between helicase and topoisomerase activities has been previously proposed to facilitate aspects of DNA replication or recombination in both Bacteria and Eukarya. Our results suggest a general role of the association of such activities in maintaining genome integrity and a mutual effect of DNA topology and repair. The abbreviations used are: Topo, topoisomerase; CCA, covalent complex assay; MES, 4-morpholineethanesulfonic acid.

Research paper thumbnail of Hyperthermophilic Archaea as model systems to study origin and evolution of early organisms

Exo- …, 2002

The current preponderance of geological and geochemical evidence favours a warm to hot Earth duri... more The current preponderance of geological and geochemical evidence favours a warm to hot Earth during the first few hundred million years after accretion. Nowadays, volcanic areas, essentially unchanged for at least 4.3 Ga, are populated by hyperthermophilic microorganisms, the ...

Research paper thumbnail of In vitro studies of DNA mismatch repair proteins

Analytical Biochemistry, 2011

The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells i... more The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells is important for understanding mechanisms involved in mutagenesis and tumorigenesis. Since mismatch repair proteins recognize mismatches containing both normal and chemically altered or damaged bases, in vitro assays must accommodate a variety of mismatches in different sequence contexts. Here we describe the construction of DNA mismatch substrates containing G:T or O 6 meG:T mismatches, the purification of recombinant native human MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) proteins, and in vitro mismatch repair and excision assays that can be adapted to study mismatch repair in nuclear extracts from mismatch repair proficient and deficient cells.

Research paper thumbnail of The mismatch repair-mediated cell cycle checkpoint response to fluorodeoxyuridine

Journal of Cellular Biochemistry, Jun 9, 2008

The loss of DNA mismatch repair (MMR) is responsible for hereditary nonpolyposis colorectal cance... more The loss of DNA mismatch repair (MMR) is responsible for hereditary nonpolyposis colorectal cancer and a subset of sporadic tumors. Acquired resistance or tolerance to some anti-cancer drugs occurs when MMR function is impaired. 5-Fluorouracil (FU), an anti-cancer drug used in the treatment of advanced colorectal and other cancers, and its metabolites are incorporated into RNA and DNA and inhibit thymidylate synthase resulting in depletion of dTTP and incorporation in DNA of uracil. Although the MMR deficiency has been implicated in tolerance to FU, the mechanism of cell killing remains unclear. Here, we examine the cellular response to fluorodeoxyuridine (FdU) and the role of the MMR system. After brief exposure of cells to low doses of FdU, MMR mediates DNA damage signaling during S-phase and triggers arrest in G2/M in the first cell cycle in a manner requiring MutSα, MutLα, and DNA replication. Cell cycle arrest is mediated by ATR kinase and results in phosphorylation of Chk1 and SMC1. MutSα binds FdU:G mispairs in vitro consistent with its being a DNA damage sensor. Prolonged treatment with FdU results in an irreversible arrest in G2 that is independent of MMR status and leads to the accumulation of DNA lesions that are targeted by the base excision repair (BER) pathway. Thus, MMR can act as a direct sensor of FdU-mediated DNA lesions eliciting cell cycle arrest via the ATR/Chk1 pathway. However, at higher levels of damage, other damage surveillance pathways such as BER also play important roles.

Research paper thumbnail of In vitro studies of DNA mismatch repair proteins

Analytical Biochemistry, Jun 1, 2011

The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells i... more The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells is important for understanding mechanisms involved in mutagenesis and tumorigenesis. Since mismatch repair proteins recognize mismatches containing both normal and chemically altered or damaged bases, in vitro assays must accommodate a variety of mismatches in different sequence contexts. Here we describe the construction of DNA mismatch substrates containing G:T or O 6 meG:T mismatches, the purification of recombinant native human MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) proteins, and in vitro mismatch repair and excision assays that can be adapted to study mismatch repair in nuclear extracts from mismatch repair proficient and deficient cells.

Research paper thumbnail of In vitro studies of DNA mismatch repair proteins

Analytical Biochemistry, 2011

The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells i... more The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells is important for understanding mechanisms involved in mutagenesis and tumorigenesis. Since mismatch repair proteins recognize mismatches containing both normal and chemically altered or damaged bases, in vitro assays must accommodate a variety of mismatches in different sequence contexts. Here we describe the construction of DNA mismatch substrates containing G:T or O 6 meG:T mismatches, the purification of recombinant native human MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) proteins, and in vitro mismatch repair and excision assays that can be adapted to study mismatch repair in nuclear extracts from mismatch repair proficient and deficient cells.

Research paper thumbnail of Lack of Strand-specific Repair of UV-induced DNA Lesions in Three Genes of the Archaeon Sulfolobus solfataricus

Journal of Molecular Biology, 2007

In all organisms, specialized systems are devoted to repair of DNA lesions induced by exposure to... more In all organisms, specialized systems are devoted to repair of DNA lesions induced by exposure to UV light. In both Eucarya and Bacteria, UV-induced pyrimidine dimers in the transcribed strand of active genes are repaired at a faster rate compared to the non-transcribed strand and the rest of the genome. Preferential repair of transcribed strands requires the Transcription-Repair Coupling Factor in Escherichia coli and the CSA and CSB proteins in humans. These factors are needed for coupling of transcription to nucleotide excision repair (NER), a major pathway for repair of UVinduced lesions. Whereas transcription-coupled NER (TC-NER) is an evolutionary conserved process, not all active genes show preferential repair of transcribed strands. The existence of a NER pathway in the Archaea has not been demonstrated directly, yet it is suggested by the presence and properties of homologues of NER nucleases and helicases. However, none of the proteins responsible for the lesion recognition steps or for TC-NER has been found in archaeal genomes. Moreover, the kinetics of gene or strand-specific repair has never been investigated in any organism of this domain. We have analysed the kinetics of repair of UV-induced DNA damage in the transcribed and non-transcribed strands of three genes of the hyperthermophilic archaeon Sulfolobus solfataricus. We found that in all three genes the two strands are repaired with the same efficiency with each other and with the genome in general, thus providing no evidence of strand bias or transcription coupling of the repair process in the genes analysed. Further studies will be required to test the existence of a transcriptioncoupled repair pathway in other archaeal genes and to elucidate the mechanism of UV lesion recognition and repair in Archaea.

Research paper thumbnail of Lack of strand-specific repair of UV-induced DNA lesions in three genes of the archaeon Sulfolobus solfataricus

Journal of molecular biology, Jan 26, 2007

In all organisms, specialized systems are devoted to repair of DNA lesions induced by exposure to... more In all organisms, specialized systems are devoted to repair of DNA lesions induced by exposure to UV light. In both Eucarya and Bacteria, UV-induced pyrimidine dimers in the transcribed strand of active genes are repaired at a faster rate compared to the non-transcribed strand and the rest of the genome. Preferential repair of transcribed strands requires the Transcription-Repair Coupling Factor in Escherichia coli and the CSA and CSB proteins in humans. These factors are needed for coupling of transcription to nucleotide excision repair (NER), a major pathway for repair of UV-induced lesions. Whereas transcription-coupled NER (TC-NER) is an evolutionary conserved process, not all active genes show preferential repair of transcribed strands. The existence of a NER pathway in the Archaea has not been demonstrated directly, yet it is suggested by the presence and properties of homologues of NER nucleases and helicases. However, none of the proteins responsible for the lesion recogniti...

Research paper thumbnail of Cytokine Immunotherapy

Experimental and Applied Immunotherapy, 2010

... M. Nelles (*) Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada e-... more ... M. Nelles (*) Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada e-mail: mnelles@uhnres.utoronto.ca Chapter 13 Cytokine Immunotherapy Megan Nelles, Vincenzo Salerno, Yixin Xu, and Christopher J. Paige Page 2. 282 M. Nelles et al. Introduction ...

Research paper thumbnail of Functional interaction of reverse gyrase with single-strand binding protein of the archaeon Sulfolobus

Nucleic Acids Research, 2005

Reverse gyrase is a unique hyperthermophilespecific DNA topoisomerase that induces positive super... more Reverse gyrase is a unique hyperthermophilespecific DNA topoisomerase that induces positive supercoiling. It is a modular enzyme composed of a topoisomerase IA and a helicase domain, which cooperate in the ATP-dependent positive supercoiling reaction. Although its physiological function has not been determined, it can be hypothesized that, like the topoisomerase-helicase complexes found in every organism, reverse gyrase might participate in different DNA transactions mediated by multiprotein complexes. Here, we show that reverse gyrase activity is stimulated by the single-strand binding protein (SSB) from the archaeon Sulfolobus solfataricus. Using a combination of in vitro assays we analysed each step of the complex reverse gyrase reaction. SSB stimulates all the steps of the reaction: binding to DNA, DNA cleavage, strand passage and ligation. By co-immunoprecipitation of cell extracts we show that reverse gyrase and SSB assemble a complex in the presence of DNA, but do not make stable protein-protein interactions. In addition, SSB stimulates reverse gyrase positive supercoiling activity on DNA templates associated with the chromatin protein Sul7d. Furthermore, SSB enhances binding and cleavage of UV-irradiated substrates by reverse gyrase. The results shown here suggest that these functional interactions may have biological relevance and that the interplay of different DNA binding proteins might modulate reverse gyrase activity in DNA metabolic pathways.

Research paper thumbnail of Transcriptional response to DNA damage in the archaeon Sulfolobus solfataricus

Nucleic Acids Research, 2003

Exposure of cells to DNA-damaging agents triggers a complex biological response involving cell cy... more Exposure of cells to DNA-damaging agents triggers a complex biological response involving cell cycle arrest and modulation of gene expression. Genomic sequencing has revealed the presence of archaeal genes homologous to components of the eucaryal nucleotide excision repair (NER) pathway, which is involved in the repair of ultraviolet (UV) lightinduced DNA damage. However, the events involved in the cell response to UV irradiation and their regulation have not been studied in Archaea. We show here that UV radiation induces the formation of cyclobutane pyrimidine dimers (CPDs) in the hyperthermophilic archaeon Sulfolobus solfataricus, and that these lesions are ef®ciently repaired in vivo in the dark, suggesting that a NER pathway is active. DNA damage is a signal for concomitant growth arrest and transcriptional induction of the NER genes XPF, XPG and XPB. The cell response to UV irradiation includes transcriptional regulation of genes encoding two DNA binding proteins involved in chromosome dynamics. Moreover, several of these genes are also strongly induced by the intercalating agent actinomycin D. Thus, response to DNA damage in S.solfataricus has features essentially conserved in all three domains of life.

Research paper thumbnail of Lack of Strand-specific Repair of UV-induced DNA Lesions in Three Genes of the Archaeon Sulfolobus solfataricus

Journal of Molecular Biology, 2007

In all organisms, specialized systems are devoted to repair of DNA lesions induced by exposure to... more In all organisms, specialized systems are devoted to repair of DNA lesions induced by exposure to UV light. In both Eucarya and Bacteria, UV-induced pyrimidine dimers in the transcribed strand of active genes are repaired at a faster rate compared to the non-transcribed strand and the rest of the genome. Preferential repair of transcribed strands requires the Transcription-Repair Coupling Factor in Escherichia coli and the CSA and CSB proteins in humans. These factors are needed for coupling of transcription to nucleotide excision repair (NER), a major pathway for repair of UV-induced lesions. Whereas transcription-coupled NER (TC-NER) is an evolutionary conserved process, not all active genes show preferential repair of transcribed strands. The existence of a NER pathway in the Archaea has not been demonstrated directly, yet it is suggested by the presence and properties of homologues of NER nucleases and helicases. However, none of the proteins responsible for the lesion recognition steps or for TC-NER has been found in archaeal genomes. Moreover, the kinetics of gene or strand-specific repair has never been investigated in any organism of this domain. We have analysed the kinetics of repair of UV-induced DNA damage in the transcribed and non-transcribed strands of three genes of the hyperthermophilic archaeon Sulfolobus solfataricus. We found that in all three genes the two strands are repaired with the same efficiency with each other and with the genome in general, thus providing no evidence of strand bias or transcription coupling of the repair process in the genes analysed. Further studies will be required to test the existence of a transcription-coupled repair pathway in other archaeal genes and to elucidate the mechanism of UV lesion recognition and repair in Archaea.

Research paper thumbnail of The mismatch repair-mediated cell cycle checkpoint response to fluorodeoxyuridine

Journal of Cellular Biochemistry, 2008

The loss of DNA mismatch repair (MMR) is responsible for hereditary nonpolyposis colorectal cance... more The loss of DNA mismatch repair (MMR) is responsible for hereditary nonpolyposis colorectal cancer and a subset of sporadic tumors. Acquired resistance or tolerance to some anti-cancer drugs occurs when MMR function is impaired. 5-Fluorouracil (FU), an anti-cancer drug used in the treatment of advanced colorectal and other cancers, and its metabolites are incorporated into RNA and DNA and inhibit thymidylate synthase resulting in depletion of dTTP and incorporation in DNA of uracil. Although the MMR deficiency has been implicated in tolerance to FU, the mechanism of cell killing remains unclear. Here, we examine the cellular response to fluorodeoxyuridine (FdU) and the role of the MMR system. After brief exposure of cells to low doses of FdU, MMR mediates DNA damage signaling during S-phase and triggers arrest in G2/M in the first cell cycle in a manner requiring MutSα, MutLα, and DNA replication. Cell cycle arrest is mediated by ATR kinase and results in phosphorylation of Chk1 and SMC1. MutSα binds FdU:G mispairs in vitro consistent with its being a DNA damage sensor. Prolonged treatment with FdU results in an irreversible arrest in G2 that is independent of MMR status and leads to the accumulation of DNA lesions that are targeted by the base excision repair (BER) pathway. Thus, MMR can act as a direct sensor of FdU-mediated DNA lesions eliciting cell cycle arrest via the ATR/Chk1 pathway. However, at higher levels of damage, other damage surveillance pathways such as BER also play important roles.

Research paper thumbnail of Reverse Gyrase Recruitment to DNA after UV Light Irradiation in Sulfolobus solfataricus

Journal of Biological Chemistry, 2004

Induction of DNA damage triggers a complex biological response concerning not only repair systems... more Induction of DNA damage triggers a complex biological response concerning not only repair systems but also virtually every cell function. DNA topoisomerases regulate the level of DNA supercoiling in all DNA transactions. Reverse gyrase is a peculiar DNA topoisomerase, specific to hyperthermophilic microorganisms, which contains a helicase and a topoisomerase IA domain that has the unique ability to introduce positive supercoiling into DNA molecules. We show here that reverse gyrase of the archaean Sulfolobus solfataricus is mobilized to DNA in vivo after UV irradiation. The enzyme, either purified or in cell extracts, forms stable covalent complexes with UV-damaged DNA in vitro. We also show that the reverse gyrase translocation to DNA in vivo and the stabilization of covalent complexes in vitro are specific effects of UV light irradiation and do not occur with the intercalating agent actinomycin D. Our results suggest that reverse gyrase might participate, directly or indirectly, in the cell response to UV light-induced DNA damage. This is the first direct evidence of the recruitment of a topoisomerase IA enzyme to DNA after the induction of DNA damage. The interaction between helicase and topoisomerase activities has been previously proposed to facilitate aspects of DNA replication or recombination in both Bacteria and Eukarya. Our results suggest a general role of the association of such activities in maintaining genome integrity and a mutual effect of DNA topology and repair. The abbreviations used are: Topo, topoisomerase; CCA, covalent complex assay; MES, 4-morpholineethanesulfonic acid.

Research paper thumbnail of Hyperthermophilic Archaea as model systems to study origin and evolution of early organisms

Exo- …, 2002

The current preponderance of geological and geochemical evidence favours a warm to hot Earth duri... more The current preponderance of geological and geochemical evidence favours a warm to hot Earth during the first few hundred million years after accretion. Nowadays, volcanic areas, essentially unchanged for at least 4.3 Ga, are populated by hyperthermophilic microorganisms, the ...

Research paper thumbnail of In vitro studies of DNA mismatch repair proteins

Analytical Biochemistry, 2011

The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells i... more The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells is important for understanding mechanisms involved in mutagenesis and tumorigenesis. Since mismatch repair proteins recognize mismatches containing both normal and chemically altered or damaged bases, in vitro assays must accommodate a variety of mismatches in different sequence contexts. Here we describe the construction of DNA mismatch substrates containing G:T or O 6 meG:T mismatches, the purification of recombinant native human MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) proteins, and in vitro mismatch repair and excision assays that can be adapted to study mismatch repair in nuclear extracts from mismatch repair proficient and deficient cells.