Functional studies of the small subunit of EcoHK31I DNA methyltransferase

Abstract

EcoHK31I DNA methyltransferase recognizes the sequence 5'-YGGCCR-3' and adds a methyl group to the fifth position of the internal cytosine to protect the DNA from cleavage by its cognate endonuclease. M.EcoHK31I is composed of polypeptides alpha and beta. Polypeptide beta only contains the conserved IX motif of the C5-MTase family, and provides a unique example to show that this motif alone may be dislocated to another polypeptide. By electromobility shift assay, we found that the alpha/beta complex recognizes specific oligonucleotide substrates. Polypeptide alpha formed aggregates with DNA, while polypeptide beta alone did not bind DNA. Therefore, polypeptide beta assists in the proper binding of polypeptide alpha to DNA substrate. The complex of polypeptide alpha and a polypeptide beta variant with an N-terminal deletion of 41 amino acids showed a 16-fold reduction in methylation activity. Further deletion resulted in an inactive methyltransferase. The dissociation equilibrium constant (Kd) of the alpha/beta complex was 56.4 nM, while the Kd value for the alpha/deltaN46-polypeptide beta complex was increased approximately 95-fold, caused by a drastic decrease in dissociate rate constant (kd) and an increase in the association rate constant (ka). This indicates that the N-terminal region of polypeptide beta takes part in subunit interaction, while the C-terminal region is involved in DNA binding.

Heterodimeric DNA methyltransferases as a platform for creating designer zinc finger methyltransferases for targeted DNA methylation in cells

“…However, this gene still conferred some methylation activity to cells, since partial protection from NotI digestion of the plasmid was observed. Since the β fragment does not require the first 42 N-terminal amino acids for activity in vitro (17), we suspected that methionine codons at positions 17 or 26 in the β fragment reading frame were serving as internal translation initiation sites. A gene in which these two sites were removed by silently mutating in the α reading frame no longer conferred any methylation activity as judged by a lack of protection at the NotI site (Supplementary Figure S2B, Supplementary Data).…”

“…In order to engineer our desired site-specific methyltransferase, we required mutants of M.EcoHK31I that can no longer assemble into a functional methyltransferase at cellular concentrations. Studies on purified M.EcoHK31I have shown that progressive deletion of amino acids from the N-terminus of the β fragment results in a progressive decrease in the association constant of the two fragments (17). This correlates with a loss of methylation activity as demonstrated by in vitro methylation assays.…”

“…This correlates with a loss of methylation activity as demonstrated by in vitro methylation assays. Almost no activity was observed when 41 amino acids were deleted from the β fragment and the protein was assayed at 1 µM concentration (the K d for fragment interaction was 2.4 µM) (17). Deletions of 42, 46 ( K d = 5.3 µM) and 50 ( K d = 7.1 µM) amino acids showed no activity at this same concentration (17).…”

“…The β chain is encoded entirely within a different reading frame of the α fragment. The large α fragment contains both the DNA binding domain and the catalytic domain (motifs I–VIII and X and TRD), whereas the smaller β fragment contains motif IX that helps stabilize site-specific binding (17). M.EcoHK31I recognizes the sequence 5′-YGGCCR-3′ and methylates the innermost cytosine.…”

“…Although M.EcoHK31I does not methylate CpG sequences, it is an attractive model protein for testing our strategy. Previous in vitro studies of M.EcoHK31I have shown that deletions of at least 42-amino acid residues from the N-terminus of the β fragment eliminated methylation activity due to a decrease in the association of the two fragments (17). Furthermore, there is growing evidence of the occurrence of non-CpG methylation in mammalian genomes of diseased or cancerous cells (18,19) and embryonic stem cells (20).…”