DNA Transposition, DNA Repair, Genome Organization
Mu transposition is mechanistically
similar to HIV integration
|Mu is a transposable bacterial virus, which propagates itself by repeated transposition/integration into the genome of its host. Cancer-causing retroviruses such as HIV use a similar mechanism to integrate into their host genomes. In the figure below, note the similarities between the Mu and HIV systems: Both genomes are flanked by the terminal dinucleotide CA which is cleaved to yield 3' OH groups. The strand transfer or joining step is identical in both systems and leads to integration of the viral genome into the host (target) genome to produce a θ intermediate. The chemistry of this class of phosphoryl transfer reactions is common to immunoglobulin rearrangements and RNA splicing.Unlike known transposable elements, which move by some one particular mechanism, Mu transposes by two alternative pathways - replicative or non-replicative transposition - depending on the phase of its life cycle. Both pathways go through a common θ intermediate, but process this intermediate differently. During integration of infecting Mu, the θ intermediate is repaired without replication, while during lytic growth it is resolved by replication. In the non-replicative repair pathway, the flanking DNA or 5' flaps attached to the ends of Mu are removed. We have discovered that the MuA transposase itself is responsible for flap removal. The flap endonuclease activity is encoded in the C-terminal domain of MuA, distinct from the DDE domain that carries out the cleavage and strand transfer steps of transposition. This activity is normally masked in the full-length protein, and has only been observed in vivo. Flap removal requires the host ClpX protein, which is known to interact with the C-terminus of MuA to remodel the transpososome for replication. We hypothesize that ClpX constitutes part of a highly regulated mechanism that unmasks the cryptic endonuclease activity of MuA specifically in the repair pathway. We have begun to actively study this pathway, which is also used by HIV, and is currently completely unexplored.|
The Mu transpososome is assembled by interactions of transposase MuA subunits with the left and right ends of Mu and an enhancer located in between. We have determined the path of the DNA within the three-site LER synapse using a method we call 'difference topology'. As shown below, the three sites (enhancer is red and the two Mu ends are blue) interwrap around each other five times. This is the most complex DNA arrangement seen to date within a recombination synapse. We are currently exploring the role of this synapse in regulation of the replication/repair decision.
|Transposition is a double-edged sword, allowing elements to populate new sites within their host genomes while potentially exposing their own DNA to self-disruption. This is a specially vexing problem for Mu because nearly half the host genome is composed of Mu sequences by the end of the lytic cycle. How does Mu avoid transposing into itself, particularly since it lacks target specificity? We have discovered a new immunity mechanism we call ‘Mu genome immunity’ that protects actively replicating/transposing Mu from self-integration. This mechanism employs strong binding of MuB throughout the Mu genome. This finding is surprising because MuB is a target-capture protein that promotes Mu integration. Our data suggest that MuB has dual and paradoxical DNA-binding properties. Based on our data, we have proposed that the replicating Mu genome is segregated into an independent ‘Mu domain’, whose formation is aided by specific Mu sequences and nucleoid associated proteins, promoting polymerization of MuB on the genome to form a barrier against self-integration. We are currently testing this model and developing MuB as a probe for chromosome structure.|