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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 EcoR124I holoenzyme is stable complex. (A) Denaturing gel filtration elution traces of EcoR124I in the presence of different concentrations of GuHCl. As identical profiles were obtained at [GuHCl] ranging from 2 to 6 M, only a representative trace done at 3 M GuHCl is shown for clarity. (B) A 12% SDS–PAGE gel of dialyzed chromatography fractions from the profile in the presence of 1 M GuHCl shown in (A). The gel was silver stained and photographed immediately; CL, column load. (C) An agarose gel showing DNA cleavage by EcoR124I at 0°C. Reactions contained 5 nM 2R+-DNA and 20 nM active EcoR124I and were initiated by the addition of ATP. Following electrophoresis, gels were stained with ethidium bromide and photographed. (D) Gel analysis of gel filtration peaks from the reaction mid-point from a DNA cleavage assay done at 0°C. Reactions were stopped by the addition of GuHCl (1 M, final) and subjected to native gel filtration. Left, agarose and right, SDS–PAGE gel lanes of gel filtration fractions. The first two agarose lanes contain aliquots from the time course prior to loading onto the column and are presented to demonstrate reaction progress. Due to the presence of 1 M GuHCl, the enzyme dissociates from the DNA and is detected only in the second peak of the elution profile. The apex fraction eluted at 15–15.3 ml and the tail fraction eluted at 16.2 ml (compared to panel B). The values to the right of the SDS–PAGE gel lanes indicate the ratio of each subunit as determined by densitometry with the value for the S-subunit being set to 1 as described previously (11). (E) Gel analysis of the reaction end-point. The reaction mix was loaded directly onto the column. Left, agarose and right, SDS–PAGE gel lanes from fractions from native gel filtration. The first two agarose lanes contain aliquots from the time course prior to loading onto the column and are presented to demonstrate reaction progress. As an excess of enzyme was used, it is detected in both the complex and free protein peaks. The values to the right of the SDS–PAGE gel lanes indicate the ratio of each subunit as determined by densitometry with the value for the S-subunit being set to 1 (11).
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Figure 3: The EcoR124I holoenzyme is stable complex. (A) Denaturing gel filtration elution traces of EcoR124I in the presence of different concentrations of GuHCl. As identical profiles were obtained at [GuHCl] ranging from 2 to 6 M, only a representative trace done at 3 M GuHCl is shown for clarity. (B) A 12% SDS–PAGE gel of dialyzed chromatography fractions from the profile in the presence of 1 M GuHCl shown in (A). The gel was silver stained and photographed immediately; CL, column load. (C) An agarose gel showing DNA cleavage by EcoR124I at 0°C. Reactions contained 5 nM 2R+-DNA and 20 nM active EcoR124I and were initiated by the addition of ATP. Following electrophoresis, gels were stained with ethidium bromide and photographed. (D) Gel analysis of gel filtration peaks from the reaction mid-point from a DNA cleavage assay done at 0°C. Reactions were stopped by the addition of GuHCl (1 M, final) and subjected to native gel filtration. Left, agarose and right, SDS–PAGE gel lanes of gel filtration fractions. The first two agarose lanes contain aliquots from the time course prior to loading onto the column and are presented to demonstrate reaction progress. Due to the presence of 1 M GuHCl, the enzyme dissociates from the DNA and is detected only in the second peak of the elution profile. The apex fraction eluted at 15–15.3 ml and the tail fraction eluted at 16.2 ml (compared to panel B). The values to the right of the SDS–PAGE gel lanes indicate the ratio of each subunit as determined by densitometry with the value for the S-subunit being set to 1 as described previously (11). (E) Gel analysis of the reaction end-point. The reaction mix was loaded directly onto the column. Left, agarose and right, SDS–PAGE gel lanes from fractions from native gel filtration. The first two agarose lanes contain aliquots from the time course prior to loading onto the column and are presented to demonstrate reaction progress. As an excess of enzyme was used, it is detected in both the complex and free protein peaks. The values to the right of the SDS–PAGE gel lanes indicate the ratio of each subunit as determined by densitometry with the value for the S-subunit being set to 1 (11).

Mentions: To address this issue, purified holoenzyme was mixed with various concentrations of GuHCl ranging from 0.5 to 6M in separate experiments, and subjected to gel filtration under conditions where the column was equilibrated in running buffer containing the same concentrations of GuHCl. Then, samples were dialyzed and subjected to electrophoresis in SDS-PAGE gels followed by analysis to ascertain the subunit composition of each peak. Results show that concentrations of GuHCl ranging from 2 to 6M dissociate the enzyme into its component subunits producing an elution profile consisting of two peaks. The first contains HsdR, while the second contains HsdM and S (Figure 3A and Supplementary Figure 1A). As the concentration of GuHCl is lowered to 1M and below, the enzyme elutes as a single peak with an apparent MW larger than that of the individual subunits alone, suggesting that it is no longer fully dissociated into its component subunits (Figure 3A). Gel analysis of the peak reveals that in 1 M GuHCl the enzyme is a mixture of free M and R subunits (eluting in the leading edge of the peak), holoenzyme (central fractions) and a complex with an R1M2S1 subunit ratio eluting in the trailing edge of the peak (Figure 3B). Loss of one R-subunit from the holoenzyme is consistent with previous work showing that the two R-subunits bind the MTase with different affinities (25).Figure 3.


Type I restriction endonucleases are true catalytic enzymes.

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

The EcoR124I holoenzyme is stable complex. (A) Denaturing gel filtration elution traces of EcoR124I in the presence of different concentrations of GuHCl. As identical profiles were obtained at [GuHCl] ranging from 2 to 6 M, only a representative trace done at 3 M GuHCl is shown for clarity. (B) A 12% SDS–PAGE gel of dialyzed chromatography fractions from the profile in the presence of 1 M GuHCl shown in (A). The gel was silver stained and photographed immediately; CL, column load. (C) An agarose gel showing DNA cleavage by EcoR124I at 0°C. Reactions contained 5 nM 2R+-DNA and 20 nM active EcoR124I and were initiated by the addition of ATP. Following electrophoresis, gels were stained with ethidium bromide and photographed. (D) Gel analysis of gel filtration peaks from the reaction mid-point from a DNA cleavage assay done at 0°C. Reactions were stopped by the addition of GuHCl (1 M, final) and subjected to native gel filtration. Left, agarose and right, SDS–PAGE gel lanes of gel filtration fractions. The first two agarose lanes contain aliquots from the time course prior to loading onto the column and are presented to demonstrate reaction progress. Due to the presence of 1 M GuHCl, the enzyme dissociates from the DNA and is detected only in the second peak of the elution profile. The apex fraction eluted at 15–15.3 ml and the tail fraction eluted at 16.2 ml (compared to panel B). The values to the right of the SDS–PAGE gel lanes indicate the ratio of each subunit as determined by densitometry with the value for the S-subunit being set to 1 as described previously (11). (E) Gel analysis of the reaction end-point. The reaction mix was loaded directly onto the column. Left, agarose and right, SDS–PAGE gel lanes from fractions from native gel filtration. The first two agarose lanes contain aliquots from the time course prior to loading onto the column and are presented to demonstrate reaction progress. As an excess of enzyme was used, it is detected in both the complex and free protein peaks. The values to the right of the SDS–PAGE gel lanes indicate the ratio of each subunit as determined by densitometry with the value for the S-subunit being set to 1 (11).
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Figure 3: The EcoR124I holoenzyme is stable complex. (A) Denaturing gel filtration elution traces of EcoR124I in the presence of different concentrations of GuHCl. As identical profiles were obtained at [GuHCl] ranging from 2 to 6 M, only a representative trace done at 3 M GuHCl is shown for clarity. (B) A 12% SDS–PAGE gel of dialyzed chromatography fractions from the profile in the presence of 1 M GuHCl shown in (A). The gel was silver stained and photographed immediately; CL, column load. (C) An agarose gel showing DNA cleavage by EcoR124I at 0°C. Reactions contained 5 nM 2R+-DNA and 20 nM active EcoR124I and were initiated by the addition of ATP. Following electrophoresis, gels were stained with ethidium bromide and photographed. (D) Gel analysis of gel filtration peaks from the reaction mid-point from a DNA cleavage assay done at 0°C. Reactions were stopped by the addition of GuHCl (1 M, final) and subjected to native gel filtration. Left, agarose and right, SDS–PAGE gel lanes of gel filtration fractions. The first two agarose lanes contain aliquots from the time course prior to loading onto the column and are presented to demonstrate reaction progress. Due to the presence of 1 M GuHCl, the enzyme dissociates from the DNA and is detected only in the second peak of the elution profile. The apex fraction eluted at 15–15.3 ml and the tail fraction eluted at 16.2 ml (compared to panel B). The values to the right of the SDS–PAGE gel lanes indicate the ratio of each subunit as determined by densitometry with the value for the S-subunit being set to 1 as described previously (11). (E) Gel analysis of the reaction end-point. The reaction mix was loaded directly onto the column. Left, agarose and right, SDS–PAGE gel lanes from fractions from native gel filtration. The first two agarose lanes contain aliquots from the time course prior to loading onto the column and are presented to demonstrate reaction progress. As an excess of enzyme was used, it is detected in both the complex and free protein peaks. The values to the right of the SDS–PAGE gel lanes indicate the ratio of each subunit as determined by densitometry with the value for the S-subunit being set to 1 (11).
Mentions: To address this issue, purified holoenzyme was mixed with various concentrations of GuHCl ranging from 0.5 to 6M in separate experiments, and subjected to gel filtration under conditions where the column was equilibrated in running buffer containing the same concentrations of GuHCl. Then, samples were dialyzed and subjected to electrophoresis in SDS-PAGE gels followed by analysis to ascertain the subunit composition of each peak. Results show that concentrations of GuHCl ranging from 2 to 6M dissociate the enzyme into its component subunits producing an elution profile consisting of two peaks. The first contains HsdR, while the second contains HsdM and S (Figure 3A and Supplementary Figure 1A). As the concentration of GuHCl is lowered to 1M and below, the enzyme elutes as a single peak with an apparent MW larger than that of the individual subunits alone, suggesting that it is no longer fully dissociated into its component subunits (Figure 3A). Gel analysis of the peak reveals that in 1 M GuHCl the enzyme is a mixture of free M and R subunits (eluting in the leading edge of the peak), holoenzyme (central fractions) and a complex with an R1M2S1 subunit ratio eluting in the trailing edge of the peak (Figure 3B). Loss of one R-subunit from the holoenzyme is consistent with previous work showing that the two R-subunits bind the MTase with different affinities (25).Figure 3.

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