Base flipping: still some unanswered questions?

It is now 20 years since the original paper by Xiaodong Cheng, Rich Roberts and colleagues was published: it remains a landmark in our understanding of the way in which nucleic acid modifying enzymes access target bases. Until that time, target bases appeared to be stably satisfied by Watson and Crick base pairing. The subsequent analysis of C5 MTase open reading frames by many labs, including those of Roberts, Bestor, Wilson, Trautner, Jeltsch etc., clearly defined the "layout" of C5MTases in which a set of relatively short motifs (I-X) combined with a Target Recognition Domain (TRD) typically in between motifs VIII and IX. In the case of the multi-specific MTases from Trautner's lab, the TRD region was shown to be expanded to accommodate several DNA recognition sites (an example would be the M.SPR enzyme which recognises, EcoRII, HaeIII and MspI sites). There are also unorthodox enzymes where the sequence is fragmented reconfigured as heterodimers: look at M.AquI and M.EcoHK31I and one example in which a TRD can be accommodated at separate locations within the polypeptide chain (see M.(phi)BssHII again from Thomas Trautner's lab). Often, these oddball enzymes can help us understand the constraints on structure function relationships amongst enzymes. 

Drawing on many genome data sets, sequence alignments via BLAST, have upheld the original insights from the Roberts and Trautner labs: the catalytic domain contains motifs I-VII, with a connection to motif X, providing a bridge to the TRD (small subdomain), which interacts with motif IX (indicated by NS, left)  to promote the flipping of the correct cytosine into the "jaws" of the catalytic Cys residue (motif IV), that is poised for attack, in part thanks to the stereochemical help from the adjacent Pro residue. As you will all know, the Pro-Cys dipeptide motif is a hallmark of these enzymes.

What do we still want to know? There are a number of groups (Norbert Reich's lab is one example), who are using state of the art biophysical methods to explore the kinetic mechanism and the structural elements in DNA MTases that drive the base flipping reaction. In addition, structural groups, such as John Tainer's lab at Scripps, are developing insight into generic aspects of base flipping (among other things!) from structural and associated biochemical data. You should all be aware of the developments in this area and keep an eye on the literature. However, my own approach has been informed by the many mutants that we and others have studied (in our case with M.MspI, M.SPRI and M.HhaI in particular). In short, we know that the chemistry of methyl transfer (from SAM to the C5 of cytosine, see right) follows from the coordinated flipping of the target base into the active site and I don't think we have anything to add to this. However, this isn't an end to the story.

Perhaps because of my (slavish?) devotion to structure-function relationships in Biochemistry, it came as no surprise to me that Laila's application of the mutagenic Pho polymerase developed by Qaiser and then Sam (from an original piece of work in Bernard Connolly's lab), demonstrated that the role of the conserved motifs in the catalytic mechanism is robust (well at least the active site Cys is essential and loss of other conserved amino acids reduces activity, but rarely abolishes it). However, probably more interestingly (to me), when Laila applied the mutagenesis strategy to the TRD region, she revealed the essential nature of a large number (of unexpected) residues. This of course is now one of our major interests and I will develop this in a broader context of enzyme action in a further Blog.