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A New DNA Recognition Mechanism: Lessons from Caulobacter Cell Cycle Regulated DNA Methyltransferase

by Clay Woodcock

Abstract: 

    DNA methylation is a key epigenetic modification controlling transcriptional levels in mammals and select bacteria. Asymmetric cellular division in the bacterial model system Caulobacter crescentus mirrors mammalian cellular differentiation in that the progenitor cell produces two morphologically and physiologically distinct daughter cells. Here, we present a detailed experimental characterization of the beta class cell cycle regulated methyltransferase, CcrM, a key global regulator maintaining temporal control of this process. This work represents the first in-depth characterization of a beta class methyltransferase.

    Examining the methylation kinetics and binding properties of CcrM for both canonical and non-specific DNA substrates, we find that CcrM appears to function by a different mechanism than previously characterized DNA modifying enzymes. CcrM displays a 106 -107 discrimination for the canonical sequence 5’-GANTC-3’ over non-cognate sites with single-nucleotide modifications 5’-GANTA-3’ 5’-AANTC-3’, a bias that is three orders of magnitude greater than the previously reported average for DNA methyltransferases. Moreover, the enzyme shows robust catalytic efficiency for both double and single-stranded substrates while maintaining above average discrimination for single-stranded DNA. This is unprecedented for a DNA methyltransferase and, so far, unique to the beta sub-class. Finally, CcrM does not appear to conform to the traditional base flipping model for sequence specificity. Instead, the catalytic activity is virtually unperturbed by non-Watson-Crick base pairs in the recognition site. Based on this observation, we propose that specific hydrogen bonds to the backbone phosphates in one strand of the DNA double helix is sufficient to account for the sequence specificity of CcrM.