Abstract
We describe the isolation and characterization of Mu A variants arrested at specific steps of transposition. Mutations at 13 residues within the Mu A protein were analyzed for precise excision of Mu DNA in vivo. A subset of the defective variants (altered at Asp269, Asp294, Gly348, and Glu392) were tested in specific steps of transposition in vitro. It is possible that at least some residues of the Asp269-Asp294-Glu392 triad may have functional similarities to those of the conserved Asp-Asp-Glu motif found in several transposases and retroviral integrases. Mu A(D269V) is defective in high-order DNA-protein assembly, Mu end cleavage, and strand transfer. The assembly defect, but not the catalytic defect, can be overcome by precleavage of Mu ends. Mu A(E392A) can assemble the synaptic complex, but cannot cleave Mu ends. A mutation of Gly348 to aspartic acid within Mu A permits the uncoupling of cleavage and strand transfer activities. This mutant is completely defective in synaptic assembly and Mu end cleavage in presence of Mg2+. The assembly defect is alleviated by replacing Mg2+ with Ca2+. Some Mu end cleavage is observed with this mutant in the presence of Mn2+. When presented with precleaved Mu ends, Mu A(G348D) exhibits efficient strand transfer activity.
Highlights
We describe the isolation and characterization of Mu transposase (Mu A) variants arrested at specific steps of transposition
This study focuses on defining the role of potential active site amino acids within the presumed catalytic domain II of Mu A by making site-directed mutations in this region
Mutations That Block Mu Excision in Vivo-It is known that mutations in Mu A that affect transposition in vitro affect Mu DNA excision in vivo (Harshey and Cuneo, 1986)
Summary
Plasmids-Plasmid pRA158 contains the Mu A gene engineered to contain two new restriction sites within the coding region without altering the amino acid sequence: an EcoRV site was created by changing T ---> A (third position of a Gly codon) at nucleotide 2137, and an HpaI site by changing G --->T (third position of a Val codon) at nucleotide 2812 on the Mu DNA sequence (Harshey et al, 1985; Priess et al, 1987). Mutations obtained by directed mutagenesis were transferred to the Mu A gene within pRA158 by replacing a wild-type restriction fragment with the corresponding mutant fragment. "Me 2SO assays" were carried out using linear DNA fragments obtained by restriction enzyme digestion from a pUC19 derivative containing R1-R2 (Namgoong et al, 1994). Strain RH208 (F' pro+lac::mini-Mukanhlprolac his met Str"; Harshey and Cuneo, 1986) was transformed with pRA158 plasmids bearing wild-type and mutant alleles of Mu A. In Vitro Assays for DNA-Protein Assembly and Transposition-Formation of the uncleaved Mu end synaptic complex (type 0) in the presence of Ca 2 + ions (Mizuuchi et al, 1992) was monitored by formation of a DSP2 (dithiobis(succinimidyl propionate))-cross-linked X structure after restriction digestion as described by Surette et al (1991). Miscellaneous Methods-Bacterial transformation, isolation of plasmid DNA, restriction enzyme digestion, DNA ligation, and other miscellaneous procedures were done as described by Maniatis et al (1982)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
