Book of Abstracts: Albany 2003

category image Albany 2003
Conversation 13
Abstract Book
June 17-21 2003

Tailoring the Activity of DNA-processing Proteins via Sequence-directed DNA Looping

DNA looping plays a key role in regulation of gene activity allowing direct contacts between proteins bound to sites well separated along the DNA chain. We demonstrate (1) that DNA looping can be induced in any chosen DNA site by sequence-directed targeting the DNA double helix with pseudocomplementary peptide nucleic acids (pcPNAs) (2). This newly introduced type of PNAs carries modified bases: adenine and thymine have been substituted for 2,6-diaminopurine (D) and 2-thiouracil (sU). The D and sU nucleobases impede association of two pcPNAs while allowing a stable Watson-Crick-type pairing of pcPNAs with the DNA counterparts. Pairs of pcPNAs can target virtually any sequence on double stranded DNA via so-called double-duplex invasion mechanism.

We designed pcPNAs to mask DNA from cleavage by type IIs restriction enzyme PleI while not interfering with the enzyme binding to its DNA recognition site. Generally, type IIs restriction endonucleases bound to their recognition sites on dsDNA require dimerization for activation of complete cleavage activity. Thus pcPNA-induced bend within the otherwise stiff DNA duplex permits direct interaction between the two enzymes bound to their target sites. One of the enzyme molecules binds tightly to the correct recognition site forming a long-lived complex. The other enzyme molecule binds transiently to a slightly sequence-degenerated site located 15-45 bp apart. Such interaction results in a totally new activity of the PleI enzyme: it produces a single-stranded break (a nick) near the degenerate site. The proper interaction of proteins is only possible when the two molecules are correctly arranged on the DNA to align their dimerization interfaces. This requirement manifests itself in a 10 bp phasing of the nicking activity with the distance between the two sites reflecting the periodicity of the DNA double helix.

To conclude, pcPNAs represent an example of a sequence-unrestricted DNA-looping ligand, which modifies the activity of DNA-bound proteins over a distance (3).

Ekaterina Protozanova*
Vadim V. Demidov
Maxim D. Frank-Kamenetskii

Center for Advanced Biotechnology
Boston University
36 Cummington Street
Boston, MA 02215, USA

References and Footnotes
  1. Protozanova, E., Demidov V. V., Soldatenkov, V., Chasovskikh, S. and Frank-Kamenetskii, M. D., EMBO Reports. 3, 956-961 (2002).
  2. Lohse, J., Dahl, O. and Nielsen, P. E., Proc. Natl. Acad. Sci. USA. 96, 11804-11808 (1999).
  3. This work was supported in part by a HFSP fellowship to E.P.