Exploring the structure and function of bacterial cytosine specific DNA methyltransferases using site-directed mutagenesis.

Point mutations were engineered into the sequence of the multispecific DNA
methyltransferase (Mtase) M. SPRI in motif IX, in order to mimic the corresponding
motif IX of mono-specific Mtase. A similar approach was adopted to modify the
sequence of the monospecific enzyme M. HhaI in motifs IX and X based on the
available structure and as a consequence the enzyme regained methylation potential.
It was thought that these changes might be sufficient to enable functional exchange
of the target recognition domains (TRDs) between a mono- and a multispecific
enzyme. However, insertion of various segments of TRD region from M. SPRI into
the M. HhaI was not successful (Chapter 4). To establish whether mono- and
multispecific Mtases are incompatible in terms of sequence exchanges, a systematic
"swapping" of motifs was carried out (Chapter 5). These experiments suggested that
there are some enzyme-specific structural interactions between different subunits
within each class of Mtases.
In second half of this thesis a bacterial two-hybrid system based on the reversible
assembly of an engineered form of M. SPRI was developed (Chapter 6). However the
Mtase protein does not assemble into an active species until a DNA segment
encoding a leucine zipper motif is fused to each of the two halves. Co-transformation
of E. coli with the plasmids expressing the C-terminal and N-terminal domains
respectively resulted in the abolition of colonies on double antibiotic plates, when an
mcr strain was used as host.
High performance liquid chromatography was used to estimate the extent of
modification of plasmids indirectly. The extent of methylation at specific sequences
within a plasmid molecule was readily detected by the corresponding differential
susceptibility to digestion by specific restriction enzymes. Using this approach it
proved possible to detect different levels of activity produced by wild type and
mutant recombinant DNA Methyltransferases with sensitivity and in a semi
quantitative manner.
In order to analyse the biochemical properties of Mtase, I have developed an in vitro
translation-modification assay. Binary studies with the mutants (from Chapter 3 and
5) showed that there were no detectable sequence-specific recognition differences
between these enzymes. Taken together, these results suggest that motif IX plays a
role in general stabilisation of the enzyme core structure and has a less significant
role in DNA recognition.