Target Immunity during Mu DNA Transposition

Greene, Eric C.; Mizuuchi, Kiyoshi

doi:10.1016/s1097-2765(02)00733-5

Cited by 39 publications

(22 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2 A. Here the -DNA was tethered to the surface by one end, and the application of a hydrodynamic force was used to maintain the molecules in an extended configuration, parallel to the sample chamber surface and within the evanescent field (19,21). This experimental design allowed continual visual inspection of fluorescent proteins bound to the DNA molecules at any point along their entire contour lengths.…”

Section: Resultsmentioning

confidence: 99%

Long-distance lateral diffusion of human Rad51 on double-stranded DNA

Granéli

Yeykal

Robertson

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

165

161

View full text Add to dashboard Cite

Rad51 is the primary eukaryotic recombinase responsible for initiating DNA strand exchange during homologous recombination. Although the subject of intense study for over a decade, many molecular details of the reactions promoted by Rad51 and related recombinases remain unknown. Using total internal reflection fluorescence microscopy, we directly visualized the behavior of individual Rad51 complexes on double-stranded DNA (dsDNA) molecules suspended in an extended configuration above a lipid bilayer. Here we show that complexes of Rad51 can bind to and slide freely along the helical axis of dsDNA. Sliding is bidirectional, does not require ATP hydrolysis, and displays properties consistent with a 1D random walk driven solely by thermal diffusion. The proteins move freely on the DNA for long periods of time; however, sliding terminates and the proteins become immobile upon encountering the free end of a linear dsDNA molecule. This study provides previously uncharacterized insights into the behaviors of human Rad51, which may apply to other members of the RecA-like family of recombinases.DNA repair ͉ homologous recombination ͉ total internal reflection fluorescence microscopy T he repair of double-stranded DNA breaks by homologous recombination is essential for maintaining genome integrity in most organisms (1-3). The importance of homologous recombination is highlighted by the finding that Rad51 null mutations are lethal in mice (4). Furthermore, defects in this repair pathway are associated with a variety of human cancers (5, 6). In eukaryotes, the broken ends of chromosomes are processed by 5Ј to 3Ј exonucleases to yield long single-stranded DNA overhangs (2, 3). Rad51, a DNA-dependent ATPase, assembles onto these overhangs, forming a nucleoprotein filament that is a key intermediate in homologous recombination (1,2,7,8). The primary functions of this filament are to locate homologous sequence that can be used as a template to repair the damaged DNA strand and to initiate strand exchange (1, 7).The structure and function of the complexes formed by Rad51 and the other RecA-like recombinases are conserved throughout evolution (8, 9). In their active states, Rad51 and related recombinases form a helical filament on DNA that induces a 50% extension of the bound DNA molecule (8). The extended nucleoprotein filament is correlated with DNA recombination activity; however, these proteins also form inactive filaments with shortened pitches (Ϸ65-85 Å versus Ϸ90-130 Å) (10). Rad51 and related recombinases also form octameric rings with a central pore large enough to accommodate a double-stranded (dsDNA) molecule (11-16). These ring-like recombinase structures do not appear to be the form of the protein that is active during the strand exchange phase of homologous recombination. Although the biological role of these rings remains unknown, it has been suggested that they may function as DNA ''pumps,'' allowing the proteins to move along DNA (12, 13).Here we have developed a unique total internal reflection fluorescence micr...

show abstract

Section: Resultsmentioning

confidence: 99%

Long-distance lateral diffusion of human Rad51 on double-stranded DNA

Granéli

Yeykal

Robertson

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

165

161

View full text Add to dashboard Cite

show abstract

“…During Mu transposition, the MuB protein mediates the target capture step. Recent in vitro studies (30,39) with phage DNA demonstrate that MuB has preferred binding sites, and these are preferred transposition targets. Interestingly, MuB binds cooperatively, forming long polymers along DNA that spread from primary binding sites.…”

Section: Discussionmentioning

confidence: 99%

Microarray analysis of transposition targets in Escherichia coli : The impact of transcription

Manna

Breier

Higgins

2004

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Transposable elements have influenced the genetic and physical composition of all modern organisms. Defining how different transposons select target sites is critical for understanding the biochemical mechanism of this type of recombination and the impact of mobile genes on chromosome structure and function. Phage Mu replicates in Gram-negative bacteria using an extremely efficient transposition reaction. Replicated copies are excised from the chromosome and packaged into virus particles. Each viral genome plus several hundred base pairs of host DNA covalently attached to the prophage right end is packed into a virion. To study Mu transposition preferences, we used DNA microarray technology to measure the abundance of >4,000 Escherichia coli genes in purified Mu phage DNA. Insertion hot-and cold-spot genes were found throughout the genome, reflecting >1,000-fold variation in utilization frequency. A moderate preference was observed for genes near the origin compared to terminus of replication. Large biases were found at hot and cold spots, which often include several consecutive genes. Efficient transcription of genes had a strong negative influence on transposition. Our results indicate that local chromosome structure is more important than DNA sequence in determining Mu target-site selection.bacteriophage Mu

show abstract

“…MuB also dissociates upon interaction with MuA in trans , but the oligomeric state of MuA, for example, monomer when bound to ends versus multimer when assembled into an active transpososome, may distinguish interactions at the ends that underlie cis immunity from those that promote target capture and transposition in trans (Fig. 8B) (102–104). Support for a cis -immunity mechanism in vivo , which would remove MuB from the vicinity of the Mu genome, comes from studies using a 10 kb derivative of Mu (Mud), which was monitored for transposition into Tn10 elements placed at various distances from the Mud element on the Salmonella typhimurium chromosome (105).…”

Section: Transposition Immunitymentioning

confidence: 99%

Transposable Phage Mu

Harshey

2014

Microbiol Spectr

View full text Add to dashboard Cite

Transposable phage Mu has played a major role in elucidating the mechanism of movement of mobile DNA elements. The high efficiency of Mu transposition has facilitated a detailed biochemical dissection of the reaction mechanism, as well as of protein and DNA elements that regulate transpososome assembly and function. The deduced phosphotransfer mechanism involves in-line orientation of metal ion-activated hydroxyl groups for nucleophilic attack on reactive diester bonds, a mechanism that appears to be used by all transposable elements examined to date. A crystal structure of the Mu transpososome is available. Mu differs from all other transposable elements in encoding unique adaptations that promote its viral lifestyle. These adaptations include multiple DNA (enhancer, SGS) and protein (MuB, HU, IHF) elements that enable efficient Mu end synapsis, efficient target capture, low target specificity, immunity to transposition near or into itself, and efficient mechanisms for recruiting host repair and replication machineries to resolve transposition intermediates. MuB has multiple functions, including target capture and immunity. The SGS element promotes gyrase-mediated Mu end synapsis, and the enhancer, aided by HU and IHF, participates in directing a unique topological architecture of the Mu synapse. The function of these DNA and protein elements is important during both lysogenic and lytic phases. Enhancer properties have been exploited in the design of mini-Mu vectors for genetic engineering. Mu ends assembled into active transpososomes have been delivered directly into bacterial, yeast, and human genomes, where they integrate efficiently, and may prove useful for gene therapy.

show abstract

Target Immunity during Mu DNA Transposition

Cited by 39 publications

References 29 publications

Long-distance lateral diffusion of human Rad51 on double-stranded DNA

Long-distance lateral diffusion of human Rad51 on double-stranded DNA

Microarray analysis of transposition targets in Escherichia coli : The impact of transcription

Transposable Phage Mu

Contact Info

Product

Resources

About