6. Common intermediates for transposition

15.6 Common intermediates for transposition


Many mobile DNA elements transpose from one chromosomal location to another by a fundamentally similar mechanism. They include IS elements, prokaryotic and eukaryotic transposons, and bacteriophage Mu. Insertion of the DNA copy of retroviral RNA uses a similar mechanism (see 16 Retroviruses and retroposons). The first stages of immunoglobulin recombination also are similar (see 24 Immune diversity).




Figure 15.10 Transposition is initiated by nicking the transposon ends and target site and joining the nicked ends into a strand transfer complex.

Transposition starts with a common mechanism for joining the transposon to its target. Figure 15.10 shows that the transposon is nicked at both ends, and the target site is nicked on both strands. The nicked ends are joined crosswise to generate a covalent connection between the transposon and the target. The two ends of the transposon are brought together in this process; for simplicity in following the cleavages, the synapsis stage is shown after cleavage, but actually occurs previously.


We know most about this process for the transposition of phage Mu, which uses the process of transposition in two ways. Upon infecting a host cell, Mu integrates into the genome by nonreplicative transposition; during the ensuing lytic cycle, the number of copies is amplified by replicative transposition. Both types of transposition involve the same type of reaction between the transposon and its target, but the subsequent reactions are different (for review see Pato, 1989; Mizuuchi, 1992).


The initial manipulations of the phage DNA are performed by the MuA transposase. Three MuA-binding sites with a 22 bp consensus are located at each end of Mu DNA. L1, L2, and L3 are at the left end; R1, R2, and R3 are at the right end. A monomer of MuA can bind to each site. MuA also binds to an internal site in the phage genome. Binding of MuA at both the left and right ends and the internal site forms a complex. The role of the internal site is not clear, but it appears to be necessary for formation of the complex, but not for strand cleavage and subsequent steps.




Figure 15.11 Mu transposition passes through three stable stages. MuA transposase forms a tetramer that synapses the ends of phage Mu. Transposase subunits act in trans to nick each end of the DNA; then a second trans action joins the nicked ends to the target DNA.

Joining the Mu transposon DNA to a target site passes through the three stages illustrated in Figure 15.11. This involves only the two sites closest to each end of the transposon. MuA subunits bound to these sites form a tetramer. This achieves synapsis of the two ends of the transposon. The tetramer now functions in a way that ensures a coordinated reaction on both ends of Mu DNA. MuA has two sites for manipulating DNA, and their mode of action compels subunits of the transposase to act in trans. The consensus-binding site binds to the 22 bp sequences that constitute the L1, L2, R1, and R2 sites. The active site cleaves the Mu DNA strands at positions adjacent to the MuA-binding sites L1 and R1. But the active site cannot cleave the DNA sequence that is adjacent to the consensus sequence in the consensus Vbinding site. However, it can cleave the appropriate sequence on a different stretch of DNA.


The ends of the transposon are thus cleaved by MuA subunits acting in trans. The trans mode of action means that the monomers actually bound to L1 and R1 do not cleave the adjacent sites. One of the monomers bound to the left end nicks the site at the right end, and vice versa. (We do not know which monomer is active at this stage of the reaction.) The strand transfer reaction also occurs in trans; the monomer at L1 transfers the strand at R1, and vice versa. It could be the case that different monomers catalyze the cleavage and strand transfer reactions for a given end (Aldaz et al., 1996; Savilahti and Mizuuchi, 1996).


The product of these reactions is a strand transfer complex in which the transposon is connected to the target site through one strand at each end. The next step of the reaction differs and determines the type of transposition. The strand transfer complex can be a target for replication (leading to replicative transposition) or for repair (leading to nonreplicative transposition).


A second protein, MuB, assists the reaction. It has an influence on the choice of target sites. Mu has a preference for transposing to a target site >10 V15 kb away from the original insertion. This is called "target immunity." It is demonstrated in an in vitro reaction containing donor (Mu-containing) and target (Mu Vdeficient) plasmids, MuA and MuB proteins, E. coli HU protein, and Mg2+ and ATP. The presence of MuB and ATP restricts transposition exclusively to the target plasmid. The reason is that when MuB binds to the MuA-Mu DNA complex, MuA causes MuB to hydrolyze ATP, after which MuB is released. However, MuB binds (nonspecifically) to the target DNA, where it stimulates the recombination activity of MuA when a transposition complex forms. In effect, the prior presence of MuA "clears" MuB from the donor, thus giving a preference for transposition to the target.


We now see in the next two sections how a common structure can be a substrate for replication (leading to replicative transposition) or used directly for breakage and reunion (leadingto nonreplicative transposition).




Reviews
Mizuuchi, K. (1992). Transpositional recombination: mechanistic insights from studies of Mu and other elements. Ann. Rev. Biochem 61, 1011-1051.
Pato, M. L. (1989). Bacteriophage Mu. In Mobile DNA, Eds. Berg, D. E. and Howe, M. American Society of Microbiology, Washington DC 23-52.

Research
Aldaz, H., Schuster, E., and Baker, T. A. (1996). The interwoven architecture of the Mu transposase couples DNA synthesis to catalysis. Cell 85, 257-269.
Savilahti, H. and Mizuuchi, K. (1996). Mu transpositional recombination: donor DNA cleavage and strand transfer in trans by the Mu transpose. Cell 85, 271-280.



Genes VII
Genes VII
ISBN: B000R0CSVM
EAN: N/A
Year: 2005
Pages: 382

flylib.com © 2008-2017.
If you may any questions please contact us: flylib@qtcs.net