Flanking host sequences can exert an inhibitory effect on the cleavage step of the in vitro mu DNA strand transfer reaction.

Z Wu,G Chaconas

doi:10.1016/s0021-9258(19)50126-0

Abstract

The effect of flanking host sequences on the cleavage step of the in vitro Mu DNA strand transfer reaction was investigated. Insertion of a mini-Mu molecule into certain sites in pUC19 results in insertions that demonstrate a decreased ability to form Type 1 complexes in subsequent rounds of transposition. Similarly, changes in the flanking host sequences directly adjacent to the Mu ends by in vitro mutagenesis can also result in Type 1-deficient mini-Mu molecules. Further examination of the inhibition revealed that Type 1 deficient mini-Mu molecules are capable of forming uncut synaptic complexes at normal levels but are compromised in their ability to serve as substrates for phosphodiester bond hydrolysis at the Mu ends. This cleavage defect can be overcome by addition of the Mu B protein and ATP to the reaction. Our data suggest that one of the roles of the B protein may be to provide a mechanism whereby Mu prophages with inhibitory flanking sequences can overcome this obstacle and avoid being trapped at unproductive locations.

Highlights

(1988))is required when the superhelical density of the donor molecule is about -0.025 but is expendable at high levels of supercoiling (Surette and Chaconas, 1989)
In addition to theprotein requirements described, there are three regions of MuDNA required for the strand transfer reaction: the left end, the right end, and the transpositional
Generation of Type 1-deficient Mini-Mu Insertions in pUC19"As diagrammed in Fig. 2, a library of mini-Mu insertions inpUC19 wasconstructed using the in vitro Mu DNA strand transfer reaction

Summary

RESULTS

Generation of Type 1-deficient Mini-Mu Insertions in pUC19"As diagrammed in Fig. 2, a library of mini-Mu insertions inpUC19 wasconstructed using the in vitro Mu DNA strand transfer reaction. Generation of Type 1-deficient Mini-Mu Insertions in pUC19"As diagrammed, a library of mini-Mu insertions inpUC19 wasconstructed using the in vitro Mu DNA strand transfer reaction. The library was used to recover Type 1-deficient mini-Mu insertions by successive rounds of enrichment as follows: mini-Mu plasmid DNA was incubated with A, HU, and IHF tfoorm Type 1 complexes (see Fig. 1).The reactions were run on agarose gels,and theunreacted supercoiledDNA band was excised and used for transformation. This process was repeated until 70%of the DNA wasno longer reactive in a standard reaction. Cleavage with MZuI, which cuts twice betweenthe Mu ends, resulted in identical digests of the two libraries, indicating that no large scale DNA rearrangements occurred during the enrichment procedure

I pWZl0 pIpUUCC1199:Mini-Mu bA

CAT GAT TAC GCC A-- GGC GTA CTA ATG CGG T-- CCG

DISCUSSION