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Martin Webb group project:

DNA helicases: mechanism of coupling ATP hydrolysis to DNA translocation

DNA helicases are ubiquitous transducing enzymes that couple the free energy of ATP hydrolysis to the separation of duplex DNA into its component single strands. They are involved in a wide range of DNA transactions, including replication and repair. There are several helicases being studied here, with different biological functions.

We are investigating the biochemical mechanism by which this important DNA processing occurs, to relate coupling of this ATPase cycle to structural changes that lead to movement of the protein along the DNA. The picture shows two measurements in real time during translocation along single stranded DNA. ATP hydrolysis was measured using the phosphate sensor and movement was measured using a fluorescent DNA.

DNA unwinding

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The picture shows a schematic of DNA unwinding during plasmid replication and a measurement of this unwinding in real time using a fluorescent reagentless biosensor for the single-stranded DNA product.

Selected publications

Toseland, CP; Martinez-Senac, MM; Slatter, AF and Webb, MR (2009)
The ATPase cycle of PcrA helicase and its coupling to translocation on DNA.
Journal of Molecular Biology 392, 1020-1032 PubMed abstract
Slatter, AF; Thomas, CD and Webb, MR (2009)
PcrA helicase tightly couples ATP hydrolysis to unwinding double-stranded DNA, modulated by the initiator protein for plasmid replication, RepD.
Biochemistry 48, 6326-6334 PubMed abstract
Martinez-Senac, MM and Webb, MR (2005)
Mechanism of translocation and kinetics of DNA unwinding by the helicase RecG.
Biochemistry 44, 16967-76 PubMed abstract
Dillingham, M. S; Wigley, D B; and Webb, MR (2002)
Direct measurement of single stranded DNA translocation by PcrA helicase using the fluorescent base analogue 2-aminopurine.
Biochemistry 41, 643-651 PubMed abstract