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Title: Motifs in the HsdR subunit of EcoKI necessary for ATP-dependent DNA translocation and DNA cleavage
Author: Davies, Graham Peter
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2000
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The type I restriction enzyme EcoKI specifies DNA methyltransferase, ATPase, DNA translocase and endonuclease activities. One subunit (HsdR) of the oligomeric enzyme contributes to those activities essential for restriction. These activities are thought to involve ATP-dependent DNA translocation and DNA cleavage. Analyses of recently predicted amino acid sequences of known and putative type I restriction endonucleases indicates conservation of eight amino acid motifs in the HsdR subunit. Seven are characteristics of the DEAD-box family of proteins that comprise known, or putative, helicases whilst the eighth was identified as a putative endonuclease motif due to its similarity to the active sites of type II restriction endonucleases and other nucleases. Secondary structure predictions based on sequences alignments of HsdR sequences suggest the DEAD-box motifs reside in domains similar to the catalytic domains of DNA helicases of known structure. Mutations affecting each of the DEAD-box motifs, including a new candidate for motif IV, impair or abolish restriction activity in vivo and ATPase activity in vitro (Webb et al., 1996; Webb, 1998; Davies et al., 1998). Alteration of conserved residues in the putative endonuclease motif resulted in complete loss of restriction in vivo and endonuclease activity in vitro. Enzyme purified from two of these mutants, those with the alterations D298E and E312H, bound DNA with an EcoKI target and hydrolysed ATP at rates equivalent to wild-type on covalently-closed circular DNA with unmodified targets without introducing any nicks or breaks into the DNA. To investigate the role of conserved motifs in HsdR on DNA translocation more directly an assay based on the EcoKI-dependent entry of T7 DNA was used (Garcia and Molineux, 1999). Mutations within the seven DEAD-box motifs abolished translation activity in vivo whereas conservation mutations in the putative endonuclease motif had no significant effect on DNA translocation in vivo.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available