Transposase contacts with mu DNA ends.

A.H Zou,P.C Leung,R.M Harshey

doi:10.1016/s0021-9258(18)54949-8

A.H Zou, P.C Leung + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)54949-8

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Abstract

We have investigated the interaction between phage Mu transposase (A protein) and the ends (att sites) of Mu by chemical and nuclease protection and interference studies. These studies define a 24-base pair contact region at five of the six att sites (L1, L3 at att L and R1, R2, R3 at att R). Hydroxyl radical footprints show that the transposase binds to one face of the DNA helix and covers two consecutive major grooves. Binding specificity is achieved primarily through the major groove. Strong contacts are found with 3 guanines which are conserved at five of the sites. Two of these guanines are missing in the weakest binding site (L2) where 13 base pairs are mainly contacted. A pair of DNAase I hypersensitive sites, one on each strand, appear at the back of only one of the two contacted major grooves at most sites except at L2, and can be correlated with the degree of A protein-induced bend (Kuo, C.-F., Zou, A., Jayaram, M., Getzoff, E. D., and Harshey, R. M. (1991) EMBO J. 10, 1585-1591) at these sites. No contacts are observed for 4-5 base pairs in the vicinity of L1 and R1, where the A protein nicks DNA during transposition.

Highlights

While comparing the A protein contacts at all the att sites, nucleotides are referred to in the text according to their alignment with site L1
It has been suggested that DNase I binds across hypersensitive sitesare found on the back of the second the minor groove of the DNA helix andthatits cutting contacted major groove
This suggests that if similar changes occur at theL1 site, they of the A-binding sequences and thehydroxyl radical footprints are manifested only in the major groove

Summary

Transposase Contactswith Mu DNA Ends*

Vol 266, No 30, Issue of October 25, pp. 20476”20482,1991 Printed in U.S.A. We have investigated the interaction between phagRe2, R3) at att R (Craigie et al, 1984). An enhanced cleavage is observed at T5 Such an enhancement, indicative perhaps of an altered DNA structure upon protein binding, is not seen on the bottom strand of L1 (Fig. 1B) or at any of the otheratt sites. For R1, the site for A protein nicking at att R, no MPE.Fe(I1) cleavage protection is observed for 5 base pairs in the neighborhood of the nicking and ethanol precipitation, the samples were resuspended in 10 mM Tris-HC1 (pH 7.5), 1 mM EDTA, and used for protein binding experiments. Ethylation Interference-DNA was ethylated as described by Siebenlist and Gilbert (1980)and used for protein binding experiments strand), one can observe that these extend a few base pairs beyond either side of the MPE footprints and include the A protein nicking site.

RESULTS

Transposase Contacts with Mu DNA Ends

DISCUSSION