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Sequence specificity of isolated DNA-cytosine methylases from Shigella sonnei 47 cells
Abstract:
Five individual DNA-cytosine methylases differing in pI (isoelectric point) values are present in Shigella sonnei 47-cells. The sequence specificity of each of those was determined 'in vitro' by a highly efficient combined approach that included pyrimidine tract (isostic) analysis, identification of the immediate neighbourhood of the methylated base within the recognition sequence and the calculation method. The enzyme with pI 5.3 (MSso5.3) is the counterpart of the RSso 47 II in the Sso 47 II restriction-modification system and methylates the internal cytosine residue of the 'palindromic' 5'-C-C-N-G-G-3' sequence. The enzymes with pI 6.2 (MSso6.2) and 7.4 (MSso7.4) exhibit identical specificity upon methylation of the 'palindromic' 5'-Py-C-N-G-Pu-3' sequence, but differ in the pI values of the proteins. The enzyme with pI 4.2 (MSso4.2) recognizes the unique tetranucleotide 5'-C-C-C-C-3' sequence and methylates the second cytosine residue at the 5'-end of the sequence. The enzyme with pI 8.4 (MSso8.4) methylates the central cytosine residue within the degenerative trinucleotide 5'-(PuC)-C-C-3' sequence. MSso5.3, MSso6.2, and MSso7.4 are presumed to belong to the 'family' of sequence-specific (Eco RII-like) enzymes. These DNA-cytosine methylases are likely to be evolutionary related to Eco RII and to have undergone a sufficient genetic drift so as to recognize similar (but more degenerative) nucleotide sequences.
Insights
Five DNA-cytosine methylases in Shigella sonnei 47 were characterized. Their distinct sequence specificities were determined, revealing novel methylation patterns and potential evolutionary links to Eco RII enzymes.
Area of Science:
- Molecular Biology
- Enzymology
- Genetics
Background:
- Shigella sonnei 47 possesses five distinct DNA-cytosine methylases with varying isoelectric point (pI) values.
- Understanding the sequence specificity of these methylases is crucial for deciphering DNA modification and restriction-modification systems.
Purpose of the Study:
- To determine the precise sequence specificity of each of the five DNA-cytosine methylases from Shigella sonnei 47.
- To investigate the potential evolutionary relationships of these methylases with known enzyme families like Eco RII.
Main Methods:
- Utilized a combined approach including pyrimidine tract (isostic) analysis.
- Identified the immediate neighborhood of the methylated base within recognition sequences.
- Employed a calculation method to determine sequence specificities 'in vitro'.
Main Results:
- MSso5.3 methylates the internal cytosine in 5'-C-C-N-G-G-3'.
- MSso6.2 and MSso7.4 share specificity for 5'-Py-C-N-G-Pu-3'.
- MSso4.2 recognizes 5'-C-C-C-C-3', methylating the second cytosine.
- MSso8.4 targets the central cytosine in 5'-(PuC)-C-C-3'.
- MSso5.3, MSso6.2, and MSso7.4 are likely Eco RII-like enzymes.
Conclusions:
- The study elucidated the unique sequence specificities of five DNA-cytosine methylases in S. sonnei 47.
- These enzymes exhibit varying recognition patterns, including palindromic and degenerate sequences.
- The findings suggest an evolutionary relationship with Eco RII, indicating genetic drift leading to modified sequence recognition.