- Email: [email protected]
On proteins, DNA and topology
234
News & Comment
TRENDS in Biotechnology Vol.20 No.6 June 2002
Journal Club
On proteins, DNA and topology In living cells, genomic DNA is organized into multiple topological domains that restrict the rotation of the double helix and cause DNA supercoiling. DNA topology is important for the intranuclear DNA packaging and plays a role in the regulation of gene expression. Particular cellular proteins are involved in the maintenance and the control of topological DNA organization. In turn, DNA topology can affect the functioning of DNAprocessing proteins.That is why various aspects of the topology-mediated interrelationship between DNA and proteins attract a great deal of attention. Three interesting papers touching on this subject from different directions were published in the January and February issues of Nucleic Acids Research. Pavlov et al. report on the finding and cloning of a new DNA-binding protein isolated from hyperthermophilic archaebacterium [1].This protein, termed 7kMk, bends DNA, leading to the formation of ~140-bp nucleosomelike loops with negative constrained DNA supercoiling.The change in DNA topology is most likely to be due to a left-handed orientation of the DNA loop, similar to a histone core of eukaryotic or archaeal nucleosomes.The 7kMk–DNA complex adds
to the list of nucleoprotein structures with looped DNA configuration that are thought to be involved in DNA packaging and in transcription regulation. Bentin and Nielsen examined whether the rotary constraints within DNA topological domains affect the performance of RNA polymerase [2]. To model transcription of a torsionally constrained template in vitro, they immobilized covalently closed, circular DNA to streptavidin-coated beads using a peptide nucleic acid (PNA)–biotin conjugate, stably targeted to a specific DNA site. For synthesis of RNAs up to 900 nt, the rate of constrained transcription elongation was as high as with rotary free DNA. Preliminary data (not shown in the paper) suggested that this could also be the case for even longer RNAs.The authors therefore concluded that genes can be transcribed in vivo by some RNA polymerases with high efficiency, despite the fact that newly synthesized RNA is entangled around the template in the narrow confines imposed by DNA topology. In another study on proteins, DNA and topology, Kuhn et al. addressed the important question of whether the rolling
DNA synthesis known as RCA is inhibited by topological constraints [3]. For this, the PNA-assisted assembly of topologically linked, earring-like DNA constructs has been applied.The RCA efficiency was unaffected when the linked templates were employed.The finding that certain DNA polymerases can carry out replicative synthesis in a topologically constrained setting could have practical implications in the area of DNA diagnostics. These recent studies on the interplay of DNA topology and DNA-binding proteins will stimulate more research on the theme covering its various aspects, with a rewarding impact on molecular biotechnology. 1 Pavlov, N.A. et al. (2002) Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri. Nucleic Acids Res. 30, 685–694 2 Bentin,T. and Nielsen, P.E. (2002) In vitro transcription of a torsionally constrained template. Nucleic Acids Res. 30, 803–809 3 Kuhn, H. et al. (2002) Rolling-circle amplification under topological constraints. Nucleic Acids Res. 30, 574–580
Vadim Demidov [email protected]
Mapping the switchboard of cellular biology by mass spectrometry Protein phosphorylation is a key posttranslational modification involved in signal transduction and the regulation of cellular processes. Simplistically, phosphorylation of proteins is a switch that turns the function of proteins on and off. It can be as simple as a digital switch, in which one phosphorylation reaction can activate a protein, or it can be more subtle, acting as an analogue switch, in which increasing phosphorylation of a protein, corresponds to increasing activity. Phosphorylation is important because diseases are often due to modifications that occur on a protein, which affects the switching mechanism. Mapping the sites of phosphorylation on proteins can help us understand the switching mechanism and understand the process involved in disease. It has been possible, but often tedious, to map the site of phosphorylation on a single http://tibtech.trends.com
purified protein using mass spectrometry. The sensitivity that could be achieved by mass spectrometry allowed only the study of proteins that could be purified in significant amounts. Until now, it was only possible to map a few switches on a very large switchboard. Ficarro et al. [1] recently reported a novel mass spectrometric approach that analyzes this phosphorylation switchboard rapidly on a proteomic scale.They combined chemical derivatization with affinity purification of phosphorylated peptides. This was followed by automated mass spectrometric identification and mapping of phosphorylated peptides. Using yeast, they demonstrated that 383 phosphorylation sites could be mapped rapidly. Furthermore, they achieved a sufficient level of detection to identify rare protein phosphorylation, such as tyrosine phosphorylation in yeast and
the phosphorylation of protein with low codon bias (i.e. low abundance). ‘...this approach could be used to see the global effects of protein phosphorylation on disease.’ The paper by Ficarro et al. opens the door to the systematic mapping of the switchboard that controls cellular regulation. In particular, this approach could be used to see the global effects of protein phosphorylation on disease. 1 Ficarro, S.B. et al. (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 302–305
Daniel Figeys [email protected]
0167-7799/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
News & Comment
TRENDS in Biotechnology Vol.20 No.6 June 2002
Journal Club
On proteins, DNA and topology In living cells, genomic DNA is organized into multiple topological domains that restrict the rotation of the double helix and cause DNA supercoiling. DNA topology is important for the intranuclear DNA packaging and plays a role in the regulation of gene expression. Particular cellular proteins are involved in the maintenance and the control of topological DNA organization. In turn, DNA topology can affect the functioning of DNAprocessing proteins.That is why various aspects of the topology-mediated interrelationship between DNA and proteins attract a great deal of attention. Three interesting papers touching on this subject from different directions were published in the January and February issues of Nucleic Acids Research. Pavlov et al. report on the finding and cloning of a new DNA-binding protein isolated from hyperthermophilic archaebacterium [1].This protein, termed 7kMk, bends DNA, leading to the formation of ~140-bp nucleosomelike loops with negative constrained DNA supercoiling.The change in DNA topology is most likely to be due to a left-handed orientation of the DNA loop, similar to a histone core of eukaryotic or archaeal nucleosomes.The 7kMk–DNA complex adds
to the list of nucleoprotein structures with looped DNA configuration that are thought to be involved in DNA packaging and in transcription regulation. Bentin and Nielsen examined whether the rotary constraints within DNA topological domains affect the performance of RNA polymerase [2]. To model transcription of a torsionally constrained template in vitro, they immobilized covalently closed, circular DNA to streptavidin-coated beads using a peptide nucleic acid (PNA)–biotin conjugate, stably targeted to a specific DNA site. For synthesis of RNAs up to 900 nt, the rate of constrained transcription elongation was as high as with rotary free DNA. Preliminary data (not shown in the paper) suggested that this could also be the case for even longer RNAs.The authors therefore concluded that genes can be transcribed in vivo by some RNA polymerases with high efficiency, despite the fact that newly synthesized RNA is entangled around the template in the narrow confines imposed by DNA topology. In another study on proteins, DNA and topology, Kuhn et al. addressed the important question of whether the rolling
DNA synthesis known as RCA is inhibited by topological constraints [3]. For this, the PNA-assisted assembly of topologically linked, earring-like DNA constructs has been applied.The RCA efficiency was unaffected when the linked templates were employed.The finding that certain DNA polymerases can carry out replicative synthesis in a topologically constrained setting could have practical implications in the area of DNA diagnostics. These recent studies on the interplay of DNA topology and DNA-binding proteins will stimulate more research on the theme covering its various aspects, with a rewarding impact on molecular biotechnology. 1 Pavlov, N.A. et al. (2002) Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri. Nucleic Acids Res. 30, 685–694 2 Bentin,T. and Nielsen, P.E. (2002) In vitro transcription of a torsionally constrained template. Nucleic Acids Res. 30, 803–809 3 Kuhn, H. et al. (2002) Rolling-circle amplification under topological constraints. Nucleic Acids Res. 30, 574–580
Vadim Demidov [email protected]
Mapping the switchboard of cellular biology by mass spectrometry Protein phosphorylation is a key posttranslational modification involved in signal transduction and the regulation of cellular processes. Simplistically, phosphorylation of proteins is a switch that turns the function of proteins on and off. It can be as simple as a digital switch, in which one phosphorylation reaction can activate a protein, or it can be more subtle, acting as an analogue switch, in which increasing phosphorylation of a protein, corresponds to increasing activity. Phosphorylation is important because diseases are often due to modifications that occur on a protein, which affects the switching mechanism. Mapping the sites of phosphorylation on proteins can help us understand the switching mechanism and understand the process involved in disease. It has been possible, but often tedious, to map the site of phosphorylation on a single http://tibtech.trends.com
purified protein using mass spectrometry. The sensitivity that could be achieved by mass spectrometry allowed only the study of proteins that could be purified in significant amounts. Until now, it was only possible to map a few switches on a very large switchboard. Ficarro et al. [1] recently reported a novel mass spectrometric approach that analyzes this phosphorylation switchboard rapidly on a proteomic scale.They combined chemical derivatization with affinity purification of phosphorylated peptides. This was followed by automated mass spectrometric identification and mapping of phosphorylated peptides. Using yeast, they demonstrated that 383 phosphorylation sites could be mapped rapidly. Furthermore, they achieved a sufficient level of detection to identify rare protein phosphorylation, such as tyrosine phosphorylation in yeast and
the phosphorylation of protein with low codon bias (i.e. low abundance). ‘...this approach could be used to see the global effects of protein phosphorylation on disease.’ The paper by Ficarro et al. opens the door to the systematic mapping of the switchboard that controls cellular regulation. In particular, this approach could be used to see the global effects of protein phosphorylation on disease. 1 Ficarro, S.B. et al. (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 302–305
Daniel Figeys [email protected]
0167-7799/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.