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Type I restriction endonucleases are true catalytic enzymes.

Bianco PR, Xu C, Chi M - Nucleic Acids Res. (2009)

Bottom Line: Following restriction, the enzymes are thought to remain associated with the DNA at the target site, hydrolyzing copious amounts of ATP.As a result, for the past 35 years type I restriction endonucleases could only be loosely classified as enzymes since they functioned stoichiometrically relative to DNA.The conclusion that type I restriction enzymes are catalytic relative to DNA has important implications for the in vivo function of these previously enigmatic enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, The State University of New York at Buffalo, Buffalo, NY 14214, USA. pbianco@buffalo.edu

ABSTRACT
Type I restriction endonucleases are intriguing, multifunctional complexes that restrict DNA randomly, at sites distant from the target sequence. Restriction at distant sites is facilitated by ATP hydrolysis-dependent, translocation of double-stranded DNA towards the stationary enzyme bound at the recognition sequence. Following restriction, the enzymes are thought to remain associated with the DNA at the target site, hydrolyzing copious amounts of ATP. As a result, for the past 35 years type I restriction endonucleases could only be loosely classified as enzymes since they functioned stoichiometrically relative to DNA. To further understand enzyme mechanism, a detailed analysis of DNA cleavage by the EcoR124I holoenzyme was done. We demonstrate for the first time that type I restriction endonucleases are not stoichiometric but are instead catalytic with respect to DNA. Further, the mechanism involves formation of a dimer of holoenzymes, with each monomer bound to a target sequence and, following cleavage, each dissociates in an intact form to bind and restrict subsequent DNA molecules. Therefore, type I restriction endonucleases, like their type II counterparts, are true enzymes. The conclusion that type I restriction enzymes are catalytic relative to DNA has important implications for the in vivo function of these previously enigmatic enzymes.

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The catalytic reaction mechanism of EcoR124I. (1) The holoenzyme binds to non-specific sites on the DNA forming the initial complex. (2) ATP binds to the enzyme facilitating transfer from non-specific DNA to the target sequence (indicated in red). Hydrolysis may occur and involve either one or both R-subunits to produce the recognition complex. (3) A second recognition complex binds to the first, forming the active dimer. (4) Rapid ATP hydrolysis coupled to dsDNA translocation ensues. Loops form due to translocation of DNA towards the stationary holoenzymes bound to their target sequences (data not shown). (5) Translocation is impeded leading to sequential DNA nicking producing dsDNA breaks. (6) Holoenzymes dissociate from the restricted DNA. The subscript 2 indicates two holoenzymes; dimers are not implied. (7) Intact holoenzymes bind to either the nascent product or to a subsequent substrate DNA if present (black/red DNA). Orange ovals, R-subunits; green ovals, M-subunits and black rectangle, S-subunit.
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Figure 5: The catalytic reaction mechanism of EcoR124I. (1) The holoenzyme binds to non-specific sites on the DNA forming the initial complex. (2) ATP binds to the enzyme facilitating transfer from non-specific DNA to the target sequence (indicated in red). Hydrolysis may occur and involve either one or both R-subunits to produce the recognition complex. (3) A second recognition complex binds to the first, forming the active dimer. (4) Rapid ATP hydrolysis coupled to dsDNA translocation ensues. Loops form due to translocation of DNA towards the stationary holoenzymes bound to their target sequences (data not shown). (5) Translocation is impeded leading to sequential DNA nicking producing dsDNA breaks. (6) Holoenzymes dissociate from the restricted DNA. The subscript 2 indicates two holoenzymes; dimers are not implied. (7) Intact holoenzymes bind to either the nascent product or to a subsequent substrate DNA if present (black/red DNA). Orange ovals, R-subunits; green ovals, M-subunits and black rectangle, S-subunit.

Mentions: The low level of active enzyme is troubling since preparations are >95% homogenous and contain stoichiometric ratios of the R-, M- and S-subunits (2:2:1; data not shown and (11)). The reason for this low level of active enzyme is unknown. To determine whether additional factors might be limiting, we repeated the protein titrations relative to R+-DNA in the presence of different concentrations of Mg[OAc]2 or SAM, in separate experiments. Similarly, we also varied the concentration of Mg[OAc]2 or SAM while holding the remaining components constant. The results show that within experimental error, the fraction of active enzyme remained unaffected (data not shown). Next, the protein titration was repeated using negatively supercoiled 2R+-DNA instead of R+-DNA. We expected that activity would saturate at 46 nM total protein. Surprisingly, saturation was observed at 22.9 nM protein indicating that the level of active protein had doubled to 44% (Figure 5A). These data suggest that EcoR124I functions as a dimer of holoenzymes consistent with previous predictions for other Type I enzymes (5,30).Figure 5.


Type I restriction endonucleases are true catalytic enzymes.

Bianco PR, Xu C, Chi M - Nucleic Acids Res. (2009)

The catalytic reaction mechanism of EcoR124I. (1) The holoenzyme binds to non-specific sites on the DNA forming the initial complex. (2) ATP binds to the enzyme facilitating transfer from non-specific DNA to the target sequence (indicated in red). Hydrolysis may occur and involve either one or both R-subunits to produce the recognition complex. (3) A second recognition complex binds to the first, forming the active dimer. (4) Rapid ATP hydrolysis coupled to dsDNA translocation ensues. Loops form due to translocation of DNA towards the stationary holoenzymes bound to their target sequences (data not shown). (5) Translocation is impeded leading to sequential DNA nicking producing dsDNA breaks. (6) Holoenzymes dissociate from the restricted DNA. The subscript 2 indicates two holoenzymes; dimers are not implied. (7) Intact holoenzymes bind to either the nascent product or to a subsequent substrate DNA if present (black/red DNA). Orange ovals, R-subunits; green ovals, M-subunits and black rectangle, S-subunit.
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Related In: Results  -  Collection

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Figure 5: The catalytic reaction mechanism of EcoR124I. (1) The holoenzyme binds to non-specific sites on the DNA forming the initial complex. (2) ATP binds to the enzyme facilitating transfer from non-specific DNA to the target sequence (indicated in red). Hydrolysis may occur and involve either one or both R-subunits to produce the recognition complex. (3) A second recognition complex binds to the first, forming the active dimer. (4) Rapid ATP hydrolysis coupled to dsDNA translocation ensues. Loops form due to translocation of DNA towards the stationary holoenzymes bound to their target sequences (data not shown). (5) Translocation is impeded leading to sequential DNA nicking producing dsDNA breaks. (6) Holoenzymes dissociate from the restricted DNA. The subscript 2 indicates two holoenzymes; dimers are not implied. (7) Intact holoenzymes bind to either the nascent product or to a subsequent substrate DNA if present (black/red DNA). Orange ovals, R-subunits; green ovals, M-subunits and black rectangle, S-subunit.
Mentions: The low level of active enzyme is troubling since preparations are >95% homogenous and contain stoichiometric ratios of the R-, M- and S-subunits (2:2:1; data not shown and (11)). The reason for this low level of active enzyme is unknown. To determine whether additional factors might be limiting, we repeated the protein titrations relative to R+-DNA in the presence of different concentrations of Mg[OAc]2 or SAM, in separate experiments. Similarly, we also varied the concentration of Mg[OAc]2 or SAM while holding the remaining components constant. The results show that within experimental error, the fraction of active enzyme remained unaffected (data not shown). Next, the protein titration was repeated using negatively supercoiled 2R+-DNA instead of R+-DNA. We expected that activity would saturate at 46 nM total protein. Surprisingly, saturation was observed at 22.9 nM protein indicating that the level of active protein had doubled to 44% (Figure 5A). These data suggest that EcoR124I functions as a dimer of holoenzymes consistent with previous predictions for other Type I enzymes (5,30).Figure 5.

Bottom Line: Following restriction, the enzymes are thought to remain associated with the DNA at the target site, hydrolyzing copious amounts of ATP.As a result, for the past 35 years type I restriction endonucleases could only be loosely classified as enzymes since they functioned stoichiometrically relative to DNA.The conclusion that type I restriction enzymes are catalytic relative to DNA has important implications for the in vivo function of these previously enigmatic enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, The State University of New York at Buffalo, Buffalo, NY 14214, USA. pbianco@buffalo.edu

ABSTRACT
Type I restriction endonucleases are intriguing, multifunctional complexes that restrict DNA randomly, at sites distant from the target sequence. Restriction at distant sites is facilitated by ATP hydrolysis-dependent, translocation of double-stranded DNA towards the stationary enzyme bound at the recognition sequence. Following restriction, the enzymes are thought to remain associated with the DNA at the target site, hydrolyzing copious amounts of ATP. As a result, for the past 35 years type I restriction endonucleases could only be loosely classified as enzymes since they functioned stoichiometrically relative to DNA. To further understand enzyme mechanism, a detailed analysis of DNA cleavage by the EcoR124I holoenzyme was done. We demonstrate for the first time that type I restriction endonucleases are not stoichiometric but are instead catalytic with respect to DNA. Further, the mechanism involves formation of a dimer of holoenzymes, with each monomer bound to a target sequence and, following cleavage, each dissociates in an intact form to bind and restrict subsequent DNA molecules. Therefore, type I restriction endonucleases, like their type II counterparts, are true enzymes. The conclusion that type I restriction enzymes are catalytic relative to DNA has important implications for the in vivo function of these previously enigmatic enzymes.

Show MeSH