Dataset Information

Characterization of proteolytic fragments of bacteriophage T7 DNA ligase.

ABSTRACT: Treatment of T7 DNA ligase with a range of proteases generates two major fragments which are resistant to further digestion. These fragments, of molecular weight 16 and 26 kDa, are derived from the N- and C-termini of the protein, respectively. The presence of ATP or a non-hydrolysable analogue, ADPNP, during limited proteolysis greatly reduces the level of digestion. The N-terminal 16 kDa region of the intact T7 ligase is labelled selectively in the presence of [alpha-32P]ATP, confirming that it contains the active site lysine residue. In common with the intact enzyme, the C-terminal portion of the protein retains the ability to band shift DNA fragments of various lengths, implicating it in DNA binding. It can also inhibit ligation by the intact protein, apparently by competing for target sites on DNA. We conclude that the N-terminal region, which contains the putative active site lysine, plays a role in the transfer of AMP from the enzyme-adenylate complex to the 5'phosphate at the nick site, while the C-terminal 26 kDa fragment appears to position the enzyme at the target site on DNA.

SUBMITTER: Doherty AJ

PROVIDER: S-EPMC145956 | biostudies-other | 1996 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Characterization of proteolytic fragments of bacteriophage T7 DNA ligase.

Doherty A J AJ Ashford S R SR Wigley D B DB

Nucleic acids research 19960601 12

Treatment of T7 DNA ligase with a range of proteases generates two major fragments which are resistant to further digestion. These fragments, of molecular weight 16 and 26 kDa, are derived from the N- and C-termini of the protein, respectively. The presence of ATP or a non-hydrolysable analogue, ADPNP, during limited proteolysis greatly reduces the level of digestion. The N-terminal 16 kDa region of the intact T7 ligase is labelled selectively in the presence of [alpha-32P]ATP, confirming that i ...[more]

PMID: 8710497

Similar Datasets

Project description:Background:The comparatively small genome, well elucidated functional genomics and rapid life cycle confer T7 bacteriophage with great advantages for bio-application. Genetic manipulation of T7 genome plays a key role in T7 related applications. As one of the important aspects in T7 phage genetic modification, gene knock-in refers to two main approaches including direct genetic manipulation in vitro and recombineering. Neither of these available methods are efficient enough to support the development of innovative applications capitalizing on T7 bio-system and thus there is room for novel strategies that address this issue. Integration mediated by the ?C31 integrase is one of the most robust site-specific recombination systems. ?C31 integrases with enhanced activity and specificity have been developed such that it is ideal to effectuate exogenous DNA knock-in of T7 phage with advanced ?C31 integrase. Methods:Plasmid construction was conducted by routine molecular cloning technology. The engineered T7 bacteriophages were constructed through homologous recombination with corresponding plasmids and the functional T7 phage was designated as T7?G10G11-attB. In the integration reaction, hosts with both executive plasmids (pEXM4) and donor plasmids (pMCBK) were lysed by T7?G10G11-attB. Progenies of T7 phages that integrated with pMCBK were isolated in restrict hosts and validated by sequencing. T7?G10G11-attB capacity limit was explored by another integration reactions with donor plasmids that contain exogenous DNA of various lengths. Results:T7?G10G11-attB exhibits abortive growth in restrictive hosts, and a bacterial attachment site recognized by ?C31 integrase (attB) was confirmed to be present in the T7?G10G11-attB genome via sequencing. The integration reaction demonstrated that plasmids containing the corresponding phage attachment site (attP) could be integrated into the T7?G10G11-attB genome. The candidate recombinant phage was isolated and validated to have integrated exogenous DNA. The maximum capacity of T7?G10G11-attB was explored, and it's found that insertion of exogenous DNA sequences longer than 2 kbp long can be accommodated stably. Conclusion:We advanced and established a novel approach for gene knock-in into the T7 genome using ?C31 integrase.

Dataset Information

Characterization of proteolytic fragments of bacteriophage T7 DNA ligase.

Publications

Characterization of proteolytic fragments of bacteriophage T7 DNA ligase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets