What is DNA helicase and its function?|How does helicase separate DNA?

                                  

                                 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.