Structural characterization of restriction/modification enzyme BsaXI complex with DNA target for structure-based design of gene-targeting reagents
EMSL Project ID
50940
Abstract
Genome engineering and targeted gene modification is a rapidly maturing discipline in which genomes within cell lines, tissues or organisms are manipulated and altered at specified individual loci. Such approaches are now being used for a wide-variety of purposes. A variety of systems are being developed for this purpose, including site-specific DNA cleaving enzymes.These systems induce double strand breaks that are repaired via homologous recombination and/or nonhomologous end joining, resulting in targeted gene modification and disruption, respectively. Structural characterization of the DNA cleavage enzymes both with and without bound DNA provide information on specific protein-DNA interactions that are crucial for the design and selection of reagents with high specificity toward desired targets.
Bacterial R-M systems consist of a restriction endonuclease (REase) that recognizes and cleaves an unmodified nucleotide sequence, and a cognate DNA methyltransferase (MTase) that protects the host DNA from cleavage by methylating an adenine or cytosine nucleotide base within its recognition sequence, hence represents an excellent model for studying specific protein-DNA interactions. We are interested in Cryo-EM characterization of large molecular complexes of bacterial enzymes which combine all three functionalities of the bacterial Restriction-Modification system (restriction, modification and recognition) in one assemblage. The goal of our study is to understand and hopefully harness, the mechanisms by which these enzymes regulation the destruction vs the protection of foreign or host genetic materials, respectively.
We are particularly interested in BsaXI which is a large bacterial type IIB R-M complex that contains all three functionalities – RAase, MTase and Recognition domain, on a single assemblage. Our goal is to study, by cryo-EM single particle averaging, the structure of BsaXI both with and without bound DNA. The specific aims are: 1. determine the structure of DNA free BsaXI; 2. determine the structure of DNA free BsaXI with antibody against tag at specific locations, and 3. determine the structure of DNA bound BsaXI,
Project Details
Start Date
2019-07-24
End Date
2020-01-31
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members