Structural studies on the mechanism of Cre-loxP site-specific recombination.

Format:

Book

Thesis/Dissertation

Author/Creator:

Guo, Feng.

Contributor:

Van Duyne, Gregory D., advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Biophysics.

0786.

Penn dissertations--Biochemistry.

Biochemistry--Penn dissertations.

Penn dissertations--Molecular biophysics.

Molecular biophysics--Penn dissertations.

Local Subjects:

Penn dissertations--Biochemistry.

Biochemistry--Penn dissertations.

Penn dissertations--Molecular biophysics.

Molecular biophysics--Penn dissertations.

0786.

Physical Description:

273 pages

Contained In:

Dissertation Abstracts International 61-03B.

System Details:

Mode of access: World Wide Web.

text file

Summary:

The site-specific recombinases from the lambda-integrase (Int) family catalyze rearrangement between specific DNA sites. In the conserved catalytic mechanism, two pairs of single strands are exchanged sequentially and a four-way Holliday junction intermediate is generated during the reaction. The Cre recombinase encoded by bacteriophage P1 represents the simplest member of the Int family because Cre recombinase and its 34-base-pair DNA binding site, loxP, are the only elements required for efficient recombination. The Cre-loxP recombination system is widely used as a tool for genetic engineering. The catalytic mechanism of the Int family recombinases is not completely understood, partially because of the multi-step nature of the reaction. The goal of this thesis is to define a high-resolution structural framework for understanding the mechanism of the Cre-loxP site-specific recombination using X-ray crystallography. Cre recombinase was first crystallized in complex with a suicide DNA substrate. This structure was solved by a combined MIR/AS/MAD method at 2.4 A resolution. The refined model revealed a tetrameric architecture with two out of four Cre subunits covalently linked to DNA. Subsequently, the structures of Cre active-site mutants bound to the other two major DNA intermediates, the Holliday junction and the synapsed duplex, were determined by molecular replacement methods. The Holliday junction complex structures are the first examples of Holliday junctions solved at high resolution. The synapsed duplex intermediate structure revealed an asymmetric kink of the DNA substrate located two base pairs away from the center of the lox site. All the intermediate structures share very similar overall architectures and suggest a recombination mechanism that does not require dramatic structural rearrangements. The structures indicate a mechanism where very subtle isomerization of the Holliday junction intermediate, rather than branch migration as proposed previously, is responsible for bridging the two strand exchange steps and for regulating the cleavage states of the active sites.

Notes:

Thesis (Ph.D. in Biochemistry and Molecular Biophysics) -- University of Pennsylvania, 2000.

Source: Dissertation Abstracts International, Volume: 61-03, Section: B, page: 1268.

Adviser: Gregory D. Van Duyne.

Local Notes:

School code: 0175.

ISBN:

9780599701014

Access Restriction:

Restricted for use by site license.