An analysis of structural and biochemical aspects of the bacterial DNA adenine-N6 methyltransferase CcrM

  • The cell-cycle regulated methyltransferase (CcrM) specifically targets the N6 position of adenine residues within GANTC DNA target sequences. Its function is essential for the viability of several members of the α-class of the proteobacteria. In Caulobacter crescentus, CcrM plays a crucial role in the control of the cell division cycle by regulating the activity of the dnaA and ctrA genes, which, in turn, control a large number of genes with functions related to cell division and differentiation. While the biological function of CcrM has been well characterized, to date very little is known about its biochemistry. In this study, we have conducted a series of experiments meant to shed more light on the functional details of CcrM-catalyzed DNA methylation. We have established that CcrM methylates DNA in a distributive manner, correcting a pre-existing erroneous view, which held that CcrM was highly processive. Furthermore, through a combined bioinformatical and biochemical approach, we have found evidence of an indirect readout mechanism employed by CcrM in target sequence recognition. Several non-catalytic amino acid residues, when mutated to alanine, led to partial or complete loss of activity of the enzyme, suggesting a role in DNA recognition. In addition, we have identified a putative domain located at the CcrM C-terminus which is moderately conserved among methyltransferases targeting GANTC sequences, but is completely absent in others. If this C-terminal domain is removed, the truncated enzyme is no longer catalytically active. When separated, both the CcrM C-terminal domain and the N-terminal portion showed a weak DNA binding ability, which suggests that the C-terminal domain has a role in DNA binding, with the involvement of both the C- and N-terminal portions being needed for strong binding through a multidentate mechanism. Finally, scanning force microscopy data revealed the ability of CcrM to simultaneously bind two DNA molecules, which suggests intersegment transfer as a mechanism for target site localization in vivo.

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Publishing Institution:IRC-Library, Information Resource Center der Jacobs University Bremen
Granting Institution:Jacobs Univ.
Author:Razvan Florin Albu
Referee:Albert Jeltsch, Susanne Illenberger, Wolfgang Hilt
Advisor:Albert Jeltsch
Persistent Identifier (URN):urn:nbn:de:101:1-201305294831
Document Type:PhD Thesis
Language:English
Date of Successful Oral Defense:2012/10/22
Year of Completion:2012
Date of First Publication:2012/12/13
PhD Degree:Biochemistry
School:SES School of Engineering and Science
Library of Congress Classification:Q Science / QD Chemistry / QD241-441 Organic chemistry / QD415-436 Biochemistry / QD431 Proteins, peptides, amino acids, etc.
Call No:Thesis 2012/44

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