Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.557530
Title: Bioinformatic and biochemical characterization of helicases from bacteriophage T5
Author: Wong, Io Nam
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2012
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Abstract:
Bacteriophage T5 is a bacterial virus known to have a remarkably high replication rate. It is a double-stranded DNA virus and encodes many of the proteins needed for its own replication. During replication, the viral double-stranded genomic DNA has to be separated by enzymes called helicases, which are motor proteins that utilize chemical energy from ATP to move along and unwind nucleic acid duplexes. Until now, no helicase has been characterized in bacteriophage T5. A bioinformatic analysis on the T5 replication gene cluster showed that several early gene products (D2, D6 and D10), which possess key helicase signature sequences (motifs), may be T5 helicases. This is the first report to investigate helicases of bacteriophage T5 and the study focused on bioinformatic and biochemical characterization of these three potential helicases. Here, D2 and D10 were identified to be two novel T5 helicases, showing helicase activity in vitro as well as having some unique properties previously uncharacterised in other helicases. However, D6 did not show ATPase activity under the condition employed and a further investigation on characteristics of D6 is required. Except for a Walker A motif, no other common conserved motifs related to helicase activity were identified in the D2 protein sequence. However, D2 was found to have a rare bipolar helicase activity giving it the ability to unwind partial duplex DNA with either a 5' or a 3' ssDNA tail (ss-dsDNA). This indicates D2 may possess some unconventional motifs relevant to its helicase activity. The extent of 5'→3' or 3'→5' unwinding activity of D2 was revealed to be dependent on 5' or 3' tail length. Interestingly, D2 displayed biased polarity preference with its 3'→5' unwinding activity being several fold greater than its 5'→3' unwinding activity when the substrates have identical tail length. Differential inhibition of the bipolar helicase activities by high NaCl concentration was also observed. The 5'→3' unwinding activity was more sensitive to inhibition by high NaCl concentration than the 3'→5' unwinding activity. The D10 protein can unwind branched DNA substrates, including forks, Y-junctions and Holliday junctions, which resemble DNA replication, recombination and repair intermediates. Furthermore, D10 was shown to catalyze branch migration of the Holliday junction substrate. Intriguingly, the ability of D10 to unwind the Y-junction substrate was found to be structure-dependent and sequence-dependent. Also, the unwinding activity can be affected by the strand discontinuity of the substrate. All the findings in this study contribute to a new insight into functional properties of helicases.
Supervisor: Sanders, Cyril Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.557530  DOI: Not available
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