Characterisation of new restriction endonucleases and molecular interaction studies with Kpnl restriction-modification system

Abstract

Characterization of New Restriction Endonucleases and Molecular Interaction Studies with the KpnI Restriction-Modification System

Abstract and Synopsis Introduction Type II restriction-modification (R-M) systems are the simplest among R-M systems. They comprise two enzymatic activities:

A site-specific nuclease (endonuclease) that recognizes and cleaves DNA at a specific recognition sequence, requiring only Mg² as a cofactor.

A methyltransferase (Mtase) that recognizes the same sequence and methylates one of the bases, using AdoMet as a cofactor. Methylated DNA is protected from cleavage by the corresponding endonuclease.

Since their discovery, extensive efforts have been made to isolate and characterize new R-M systems. Over 3000 type II systems have been identified, making them one of the largest groups of site-specific DNA-binding proteins. These enzymes serve as model systems for understanding site specificity and the role of divalent metal ions in DNA binding and catalysis.

This thesis focuses on:

Characterization of two new endonucleases (Enases).

Over-expression of the KpnI R-M system, as well as SmaI and HindIII endonucleases.

Detailed molecular interaction studies of the KpnI endonuclease and methyltransferase.

Chapter 2: Identification of New Endonucleases

Two new type II endonucleases, StiI (from Streptomyces thermodiastaticus) and SasI (from Bacillus species), were identified.

Recognition sequences:

StiI: 5'-CTCGAG-3'

SasI: 5'-CANNNNNTGG-3'

Optimum temperature, pH, and cation requirements were determined.

Chapter 3: Over-Expression of KpnI R-M System

Genes encoding KpnI endonuclease and methyltransferase from Klebsiella pneumoniae were cloned and expressed in E. coli using a two-plasmid strategy.

KpnI endonuclease was hyper-expressed in a T7 system, reaching 15-30% of cellular protein.

Both enzymes were purified to homogeneity in a single chromatographic step.

Oligomeric status: both enzymes exist as dimers in solution (unusual for Mtases, which are typically monomers).

Binding studies:

KpnI endonuclease binds its cognate site without cofactors.

KpnI Mtase binds both specific and non-specific DNA.

Chapter 4: Differential Interaction Studies

Footprinting techniques revealed:

KpnI endonuclease protects a 14-18 bp region.

KpnI Mtase protects a larger region.

Endonuclease interacts with guanine residues and phosphate groups within the recognition sequence (5'-GGTACC-3').

Mtase induces DNA distortion, evidenced by thymine hyper-reactivity to permanganate oxidation.

These differences reflect the contrasting chemistry of DNA cleavage vs. methylation.

Chapter 5: Star Activity and Metal Ion Effects

KpnI endonuclease exhibits Ca² -dependent DNA cleavage activity, unique among type II enzymes.

Ca² confers exquisite specificity, suppressing Mg² -induced promiscuous activity.

Several star sites were mapped and compared with cognate sites, confirming Ca² -dependent site-specific cleavage.

Appendix: Expression of SmaI and HindIII Endonucleases

A novel T7 promoter-based regulatory circuit was engineered for tight control.

Components: T7 lysozyme (inhibitor of T7 RNA polymerase) and a high-affinity DNA-binding activator protein.

Expression of SmaI and HindIII was observed only in induced cell extracts, demonstrating tight regulation.

Conclusions Two new restriction endonucleases (StiI and SasI) were identified and characterized.

KpnI R-M system was successfully over-expressed and purified, revealing unusual dimeric Mtase behavior.

Molecular interaction studies highlighted distinct DNA binding patterns of KpnI endonuclease and Mtase.

KpnI endonuclease shows unique Ca² -dependent cleavage specificity.

A novel regulatory circuit enabled controlled expression of toxic endonucleases (SmaI, HindIII).