Details
| Original language | English |
|---|---|
| Pages (from-to) | 5104-11 |
| Number of pages | 8 |
| Journal | The EMBO journal |
| Volume | 15 |
| Issue number | 18 |
| Publication status | Published - 16 Sept 1996 |
| Externally published | Yes |
Abstract
Linear diffusion along DNA is a mechanism of enhancing the association rates of proteins to their specific recognition sites on DNA. It has been demonstrated for several proteins in vitro, but to date in no case in vivo. Here we show that the restriction endonuclease EcoRV slides along the DNA, scanning approximately 1000 bp in one binding event. This process is critically dependent on contacts between amino acid residues of the protein and the backbone of the DNA. The disruption of single hydrogen bonds and, in particular, the alteration of electrostatic interactions between amino acid side chains of the protein and phosphate groups of the DNA interfere with or abolish effective sliding. The efficiency of linear diffusion is dependent on salt concentration, having a maximum at 50 mM NaCl. These results suggest that a nonspecific and mobile binding mode capable of linear diffusion is dependent on a subtle balance of forces governing the interaction of the enzyme and the DNA. A strong correlation between the ability of EcoRV mutants to slide along the DNA in vitro and to protect Escherichia coli cells from phage infection demonstrates that linear diffusion occurs in vivo and is essential for effective phage restriction.
Keywords
- DNA/metabolism, Deoxyribonucleases, Type II Site-Specific/metabolism, Diffusion, Models, Molecular, Polymerase Chain Reaction, Protein Conformation
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In: The EMBO journal, Vol. 15, No. 18, 16.09.1996, p. 5104-11.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Linear diffusion of the restriction endonuclease EcoRV on DNA is essential for the in vivo function of the enzyme
AU - Jeltsch, A
AU - Wenz, C
AU - Stahl, F
AU - Pingoud, A
PY - 1996/9/16
Y1 - 1996/9/16
N2 - Linear diffusion along DNA is a mechanism of enhancing the association rates of proteins to their specific recognition sites on DNA. It has been demonstrated for several proteins in vitro, but to date in no case in vivo. Here we show that the restriction endonuclease EcoRV slides along the DNA, scanning approximately 1000 bp in one binding event. This process is critically dependent on contacts between amino acid residues of the protein and the backbone of the DNA. The disruption of single hydrogen bonds and, in particular, the alteration of electrostatic interactions between amino acid side chains of the protein and phosphate groups of the DNA interfere with or abolish effective sliding. The efficiency of linear diffusion is dependent on salt concentration, having a maximum at 50 mM NaCl. These results suggest that a nonspecific and mobile binding mode capable of linear diffusion is dependent on a subtle balance of forces governing the interaction of the enzyme and the DNA. A strong correlation between the ability of EcoRV mutants to slide along the DNA in vitro and to protect Escherichia coli cells from phage infection demonstrates that linear diffusion occurs in vivo and is essential for effective phage restriction.
AB - Linear diffusion along DNA is a mechanism of enhancing the association rates of proteins to their specific recognition sites on DNA. It has been demonstrated for several proteins in vitro, but to date in no case in vivo. Here we show that the restriction endonuclease EcoRV slides along the DNA, scanning approximately 1000 bp in one binding event. This process is critically dependent on contacts between amino acid residues of the protein and the backbone of the DNA. The disruption of single hydrogen bonds and, in particular, the alteration of electrostatic interactions between amino acid side chains of the protein and phosphate groups of the DNA interfere with or abolish effective sliding. The efficiency of linear diffusion is dependent on salt concentration, having a maximum at 50 mM NaCl. These results suggest that a nonspecific and mobile binding mode capable of linear diffusion is dependent on a subtle balance of forces governing the interaction of the enzyme and the DNA. A strong correlation between the ability of EcoRV mutants to slide along the DNA in vitro and to protect Escherichia coli cells from phage infection demonstrates that linear diffusion occurs in vivo and is essential for effective phage restriction.
KW - DNA/metabolism
KW - Deoxyribonucleases, Type II Site-Specific/metabolism
KW - Diffusion
KW - Models, Molecular
KW - Polymerase Chain Reaction
KW - Protein Conformation
M3 - Article
C2 - 8890184
VL - 15
SP - 5104
EP - 5111
JO - The EMBO journal
JF - The EMBO journal
SN - 0261-4189
IS - 18
ER -