Members of the large serine recombinase (LSR) family of enzymes carry out recombination between short, specific DNA sequences commonly referred to as attP and attB. For many LSR enzymes, the reaction is effectively irreversible and does not require accessory proteins, additional DNA elements, or other cofactors. These features have led to the emergence of LSRs as important new tools that can carry out precise manipulations of eukaryotic genomes and show promise in gene therapy applications. Despite the importance of the LSRs, our current mechanistic understanding of how these enzymes function is limited. There are no structural models available for any LSR protein and no indication of how the recombinases bind to the attP and attB recombination sites. It has therefore been difficult to understand how the LSR enzymes exhibit such high site selectivity and strong directionality. To address these gaps in our understanding of LSR structure and function, we will use the bacteriophage A118 integrase as a model system to (i) test a mechanism where LSR enzymes use an unusual, extended coiled-coil motif to regulate the recombination reaction, and (ii) develop a structural and energetic framework for understanding selective association, or synapsis, of the recombining sites. Our functional model for LSR regulation of site-specificity and directionality is inspired by a A118 integrase-DNA complex structure that we have recently determined. These studies will provide an important framework for our long-term goal engineering LSRs to integrate preferentially into a single site in the human genome.

Public Health Relevance

Successful completion of the proposed research will lead to a detailed understanding of how large serine recombinases carry out site-specific recombination between DNA attachment sites with high site specificity and strong directionality. These integrase enzymes are capable of efficiently integrating transgenes into the human genome, targeting pseudo-attachment sites. This project will provide the groundwork necessary to engineer the integrase and its recombining sites for custom and novel applications in genome engineering. These tools show promise in both experimental and therapeutic applications, including human gene therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM108751-01
Application #
8612126
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Sakalian, Michael
Project Start
2014-02-01
Project End
2018-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
1
Fiscal Year
2014
Total Cost
$300,003
Indirect Cost
$110,003
Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Tong, Wenjun; Warren, David; Seah, Nicole E et al. (2014) Mapping the ? Integrase bridges in the nucleoprotein Holliday junction intermediates of viral integrative and excisive recombination. Proc Natl Acad Sci U S A 111:12366-71
Seah, Nicole E; Warren, David; Tong, Wenjun et al. (2014) Nucleoprotein architectures regulating the directionality of viral integration and excision. Proc Natl Acad Sci U S A 111:12372-7
Rutherford, Karen; Van Duyne, Gregory D (2014) The ins and outs of serine integrase site-specific recombination. Curr Opin Struct Biol 24:125-31