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How is modification of the DNA substrate recognized by the PvuII restriction endonuclease?
J R Horton1, J Bonventre, X Cheng
1Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
Abstract:
In restriction-modification systems, cleavage of substrate sites in cellular DNA by the restriction endonuclease is prevented by the action of a cognate methyltransferase that acts on the same substrate sites. The PvuII restriction endonuclease (R.PvuII) has been structurally characterized in a complex with substrate DNA (Cheng et al., 1994) and as an apoenzyme (Athanasiadis et al., 1994). We report here a structure, determined to 1.9 A resolution by crystallography, of a complex between R.PvuII and iodinated DNA. The presence of an iodine at the 5-carbon of the methylatable cytosine results in the following changes in the protein: His84 moved away from the modified base; this movement was amplified in His85 and disrupts an intersubunit hydrogen bond; and the base modification disturbs the distribution of water molecules that associate with these histidine residues and the area of the scissile bond. Considering these observations, hypotheses are given as to why a similar oligonucleotide, where a methyl group resides on the 5-carbon of the methylatable cytosine, is slowly cleaved by R.PvuII (Rice et al., 1995).
Insights
Structural analysis of PvuII restriction endonuclease (R.PvuII) reveals how DNA base modification by iodine binding affects protein structure and function. This provides insights into R.PvuII
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- Restriction-modification systems protect cellular DNA from endonuclease cleavage via methyltransferase action.
- The PvuII restriction endonuclease (R.PvuII) structure is known in complex with DNA and as an apoenzyme.
- Understanding R.PvuII-DNA interactions is crucial for DNA modification and cleavage mechanisms.
Purpose of the Study:
- To determine the crystal structure of R.PvuII in complex with iodinated DNA at 1.9 A resolution.
- To elucidate the structural consequences of 5-iodocytosine modification on R.PvuII.
- To propose mechanisms explaining R.PvuII's slow cleavage of methylated DNA.
Main Methods:
- X-ray crystallography
- Protein-DNA complex structure determination
- Structural analysis of R.PvuII-DNA interactions
Main Results:
- The structure of R.PvuII complexed with iodinated DNA was determined to 1.9 A resolution.
- Iodine at the 5-carbon of cytosine caused His84 to move away from the modified base, affecting His85 and an intersubunit hydrogen bond.
- Base modification altered water molecule distribution near histidine residues and the scissile bond area.
Conclusions:
- The structural changes induced by 5-iodocytosine modification provide a basis for understanding R.PvuII's altered DNA cleavage activity.
- Hypotheses are presented regarding the slow cleavage of methylated DNA by R.PvuII based on observed structural perturbations.
- This study enhances the understanding of restriction endonuclease mechanisms and DNA recognition.