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Proteolytic processing of the Ada protein that repairs DNA O6-methylguanine residues in E. coli
Abstract:
In extracts of E. coli treated with an adapting regime of MNNG, the induced 39kd Ada protein having O6-MeG-DNA methyltransferase activity is processed to a 19kd active domain corresponding to the C-terminal half of the intact protein. This proteolytic processing has been followed on Western immunoblots using antisera raised against the 19kd fragment. Initial processing at 25 degrees C or 37 degrees C mainly generates a fragment of mol. wt. 24kd which then undergoes a slower second cleavage to generate the 19kd active domain. Preceding this second cleavage site is a sequence of amino acids Thr- -Gly-Met-Thr- -Lys that also occurs at another site in the N-terminal half of the 39kd methyltransferase. It is proposed that this sequence is a recognition site for proteolytic activity. On the basis of cleavage of the Ada protein at either one or both of these sites, fragments may be generated of mol. wt. 24kd and 19kd containing the active site for O6-methylguanine and O4-methylthymine repair, and 15kd and 20kd, containing the active site for methylphosphotriester repair. These observations explain previous reports by others on the existence in cell extracts of multiple methyltransferase activities of different sizes recognizing O-methyl lesions in DNA. The cellular protease involved is resistant to a wide range of protease inhibitors.
Insights
The Ada protein in E. coli, responsible for DNA repair, is processed into smaller active fragments. This proteolytic processing generates distinct DNA repair methyltransferase domains of varying sizes.
Area of Science:
- Molecular Biology
- Enzymology
- DNA Repair Mechanisms
Background:
- The Ada protein in E. coli possesses O6-methylguanine-DNA methyltransferase activity.
- Adaptation to MNNG treatment induces the expression of the 39 kDa Ada protein.
Purpose of the Study:
- To investigate the proteolytic processing of the 39 kDa Ada protein.
- To characterize the active domains generated from Ada protein cleavage.
- To explain the existence of multiple DNA repair methyltransferase activities.
Main Methods:
- Western immunoblotting using antisera against the 19 kDa fragment.
- Analysis of protein fragments generated at different temperatures (25°C and 37°C).
Main Results:
- The 39 kDa Ada protein is processed to a 19 kDa active C-terminal domain.
- Initial cleavage yields a 24 kDa fragment, followed by a slower second cleavage to the 19 kDa domain.
- A specific amino acid sequence (Thr-Gly-Met-Thr-Lys) is proposed as a proteolytic recognition site.
- Cleavage generates fragments (19-24 kDa) for O6-methylguanine/O4-methylthymine repair and (15-20 kDa) for methylphosphotriester repair.
Conclusions:
- Proteolytic processing of the Ada protein explains the presence of multiple DNA repair methyltransferase activities of different molecular weights.
- The cellular protease responsible for this processing is highly resistant to inhibitors.