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Published on: October 31, 2014
Effect of mutations in the Mu-host junction region on transpososome assembly
1The Department of Biochemistry, University of Western Ontario, London, Ontario, N6A 5C1, Canada.
Abstract:
Mu transposition occurs through a series of higher-order nucleoprotein complexes called transpososomes. The region where the Mu DNA joins the host DNA plays an integral role in the assembly of these transpososomes. We have created a series of point mutations at the Mu-host junction and characterized their effect on the Mu in vitro strand transfer reaction. Analysis of these mutant constructs revealed an inhibition in transpososome assembly at the point in the reaction pathway when the junction region is engaged by the transposase active site (i.e. the transition from LER to type 0). We found that the degree of inhibition was dependent upon the particular base-pair change at each position and whether the substitution occurred at the left or right transposon end. The MuB transposition protein, an allosteric effector of MuA, was shown to suppress all of the inhibitory Mu-host junction mutants. Most of the mutant constructs were also suppressed, to varying degrees, by the substitution of Mg(2+) with Mn(2+). Analysis of the mutant constructs has revealed hierarchical nucleotide preferences at positions -1 through +3 for transpososome assembly and suggests the possibility that specific metal ion-DNA base interactions are involved in DNA recognition and transpososome assembly.
Insights
Mu transposition involves complex protein structures called transpososomes. Mutations at the Mu-host DNA junction disrupt transpososome assembly, but MuB protein and Mn(2+) can restore the process.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Bacterial insertion sequences like bacteriophage Mu utilize a complex process called transposition to move within a host genome.
- Transposition is mediated by higher-order nucleoprotein complexes known as transpososomes, which are crucial for the DNA integration process.
- The junction region between the Mu DNA and the host DNA is critical for the proper assembly of these transpososomes.
Purpose of the Study:
- To investigate the role of the Mu-host DNA junction sequence in transpososome assembly and Mu transposition.
- To characterize the effects of specific point mutations at the Mu-host junction on the in vitro strand transfer reaction.
- To identify factors that can overcome inhibitory effects caused by junction mutations.
Main Methods:
- Site-directed mutagenesis was used to create point mutations at the Mu-host junction.
- In vitro strand transfer assays were performed to analyze the effects of these mutations on transpososome assembly and function.
- The influence of MuB protein and different divalent metal ions (Mg(2+) vs. Mn(2+)) on mutant transposition activity was assessed.
Main Results:
- Mutations at the Mu-host junction significantly inhibited transpososome assembly, particularly during the transition involving transposase active site engagement.
- The degree of inhibition varied depending on the specific base-pair substitution and its location (left vs. right transposon end).
- The MuB protein fully suppressed all inhibitory junction mutants, while Mn(2+) partially suppressed most of them, indicating hierarchical nucleotide preferences at the junction site.
Conclusions:
- The Mu-host junction sequence plays a critical, position-dependent role in Mu transpososome assembly.
- Specific metal ion-DNA base interactions may be involved in the recognition of the junction DNA during transpososome formation.
- MuB protein acts as a potent suppressor of junction-mediated assembly defects, highlighting its regulatory role in Mu transposition.
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