Movement of transposable genetic elements requires the cleavage of each end of the element genome and the subsequent joining of these cleaved ends to a new target DNA site. During Mu transposition, these reactions are catalyzed by a tetramer of four identical transposase subunits bound to the paired Mu DNA ends. To elucidate the organization of active sites within this tetramer, the subunit providing the essential active site DDE residues for each cleavage and joining reaction was determined. We demonstrate that recombination of the two Mu DNA ends is catalyzed by two active sites, where one active site promotes both cleavage and joining of one Mu DNA end. This active site uses all three DDE residues from the subunit bound to the transposase binding site proximal to the cleavage site on the other Mu DNA end (catalysis in trans). In addition, we uncover evidence that the catalytic activity of these two active sites is coupled such that the coordinated joining of both Mu DNA ends is favored during recombination. On the basis of these results, we propose that the DNA joining stage requires a cooperative transition within the transposase-DNA complex. The cooperative utilization of active sites supplied in trans by Mu transposase provides an example of how mobile elements can ensure concomitant recombination of distant DNA sites.[Key Words: Transposase; DDE motif; phosphoryl transfer; active site; protein-DNA assembly; Mu transposition]Received July 27, 1999; accepted in revised form Aug 31, 1999.Transposition and retroviral integration, like all forms of site-specific recombination, require a series of DNA cleavage and joining reactions to accomplish DNA movement. The propagation of mobile genetic elements requires the spatial and sequential coordination of several DNA regions and is orchestrated by one or two proteins, called transposases or integrases, in the context of multimeric, nucleoprotein complexes. These proteins form a family related by sequence within their catalytic domains (Kulkosky et al. 1992;Polard and Chandler 1995;Rice et al. 1996). Mu transposase (MuA) is one well-studied member of this transposase/ integrase family.Multimers of transposase and retroviral integrase catalyze recombination using similar mechanisms that share two reaction steps: (1) cleavage of the element-host DNA junctions to yield a 3Ј OH at each end of the element genome and (2) joining of these two 3Ј OH ends, through single-step transesterification, to two phosphodiester bonds on opposite strands of a new DNA site (the target site) in a process called strand transfer (for review, see Mizuuchi 1992;Kleckner et al. 1995). Elements that transpose via a nonreplicative, or cut-and-paste, mechanism (e.g., Tn10, Tn7, and P elements) also cleave the 5Ј strand at each element end to excise the element completely from its old location before joining the 3Ј ends to a new DNA site (Bainton et al.
Mu transposition occurs within a large protein-DNA complex called a transpososome. This stable complex includes four subunits of MuA transposase, each contacting a 22-base pair recognition site located near an end of the transposon DNA. These MuA recognition sites are critical for assembling the transpososome. Here we report that when concentrations of Mu DNA are limited, the MuA recognition sites permit assembly of transpososomes in which non-Mu DNA substitutes for some of the Mu sequences. These "hybrid" transpososomes are stable to competitor DNA, actively transpose the non-Mu DNA, and produce transposition products that had been previously observed but not explained. The strongest activator of non-Mu transposition is a DNA fragment containing two MuA recognition sites and no cleavage site, but a shorter fragment with just one recognition site is sufficient. Based on our results, we propose that MuA recognition sites drive assembly of functional transpososomes in two complementary ways. Multiple recognition sites help physically position MuA subunits in the transpososome plus each individual site allosterically activates transposase.Transposons are found in all the biological kingdoms, and some perform specialized functions. For example, the machinery that initiates V(D)J recombination likely evolved from a transposon (1, 2), and the cDNA of HIV and other retroviruses integrate into host cell DNA through mechanisms nearly identical to transposition (3). The genome of bacteriophage Mu is a transposon that uses transposition both to integrate into the DNA of a new host cell and to replicate before lysis. Like most DNA rearrangements, transposition is a complex, multi-step process, requiring numerous DNA sequence elements. Studies of bacteriophage Mu have been central to our understanding of both the fundamental mechanisms and the complexities of DNA transposition.Phage Mu encodes a transposase, MuA, that transfers the Mu genome from one DNA location (the transposition donor) to a new location (the transposition target) (4, 5). During transposition, transposase performs two principle reactions: DNA cleavage and DNA strand transfer. During cleavage, the donor DNA is nicked twice, once at each 3Ј end of the Mu genome. During strand transfer, the cleaved transposon ends are inserted into neighboring sites on the two target strands.Little or no specific sequence information is needed on the target DNA (6), but the Mu DNA provides many sequence cues for transposition (Fig. 1). For example, the last two nucleotides at either 3Ј end of the Mu DNA, the cleavage sites, have the sequence 5Ј-CA. Also near each end of the Mu DNA are three recognition sites, distinct from the cleavage sites, which share a 22-base pair consensus sequence. The recognition sites are referred to as R1, R2, and R3 on the right end and L1, L2, and L3 on the left end ( Fig.
Telomeres are an unusual component of the genome because they do not encode genes, but their structure and cellular maintenance machinery (which we define as ''telotype'') are essential for chromosome stability. Cells can switch between different phenotypic states. One such example is when they switch from maintenance mediated by telomerase (TERT telotype) to one of the two alternative mechanisms of telomere preservation (ALT I and ALT II telotype). The nature of this switch is largely unknown. Reintroduction of telomerase into ALT II, but not ALT I, yeast led to the loss of their ability to survive a second round of telomerase withdrawal. Mating-based genetic analysis of ALT I and II revealed that both types of telomerase-independent telomere maintenance are inherited as a non-Mendelian trait dominant over senescence (SEN telotype). Additionally, inheritance of ALT I and ALT II did not depend on either the mitochondrial genome or a prion-based mechanism. Type I, but not type II, survivor cells exhibited impaired gene silencing, potentially connecting the switch to the ALT telotype epigenetic changes. These data provide evidence that nonprion epigenetic-like mechanisms confer flexibility on cells as a population to adjust to the life-threatening situation of telomerase loss, allowing cells to switch from TERT to ALT telotypes that can sustain viable populations.
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