DNA helicase
assay
Ø Helicase is the enzyme that harness
chemical energy of ATP to separate the two parental DNA strand at replicating
fork is called a helicase.
Ø Many DNA helicase have been
identified in E. coli cells
Ø The problem is in finding which of
these is involved in DNA replication.
1. Rep helicase
2. DNA helicase II
3. DNA helicase III
Ø Without a functional helicase, the
fork cannot more as DNA synthesis must had immediately.
Ø The DnaB product was known to be an
ATPase and the DnaB protein was found
associated with the primers which makes primers for DNA replication
Ø All of these finding suggested the
DnaB is the DNA helicase that unwind the DNA double helix during E.coli
DNA replication.
Ø Janathan Lebowitz and Roger Mcmacken
did an experiment in 1986.
Ø They used to helicase substrate,
which is circular M13 phage DNA, annealed to a shorter piece of linear DNA,
which was labelled at it 5` end
Ø Lebowitz and Macmacken incubated the
labelled substrate with DnaB, or other proteins, and the electrophoresed the products.
Ø If the protein has helicase activity,
it would unwind the double stranded DNA and separate the two strands.
Ø Then the short-labelled DNA would
migrate independently of the larger, unlabelled DNA, and would have a much
higher electrophoretic mobility.
Ø DnaB clone and helicase activity, ad
this was stimulated by DnaG by ssB (single stranded DNA binding protein)
Ø Neither DnaG nor ssB by themselves or
together had any helicase activity.
Ø These dnaB helicase that unwind the
DNA at the replicating fork.
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