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Interaction of Rep and DnaB on DNA
John Atkinson1, Milind K Gupta, Peter McGlynn
1School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.
Nucleic Acids Research
|October 21, 2010
Summary
Accessory helicases like Rep are crucial for DNA replication fork progression. Rep exhibits unique cooperativity with the primary helicase DnaB, particularly when single-stranded DNA is exposed on the leading strand template.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA replication requires unwinding of the template DNA by replicative helicases.
- Accessory helicases are necessary to resolve stalls caused by nucleoprotein complexes.
- In Escherichia coli, DnaB (lagging strand) and Rep (leading strand) are primary and accessory helicases, respectively.
Purpose of the Study:
- To investigate the cooperative unwinding mechanism between DnaB and Rep helicases.
- To determine the specific DNA substrate requirements for Rep-DnaB cooperativity.
- To elucidate the role of accessory helicases in overcoming replication fork obstacles.
Main Methods:
- In vitro DNA unwinding assays using model forked DNA substrates.
- Characterization of helicase-DNA interactions and complex formation.
- Analysis of cooperativity between different helicase combinations.
Main Results:
- Rep helicase displays cooperativity with DnaB, but this is not observed with other helicases.
- Cooperativity requires exposed single-stranded DNA (ssDNA) on the leading strand template.
- Rep can form stable complexes with DnaB on both linear and branched DNA substrates.
Conclusions:
- The presence of ssDNA ahead of the leading strand polymerase is critical for Rep-DnaB cooperativity at stalled replication forks.
- Rep has the flexibility to interact with ssDNA on either strand template.
- Replisome architecture, potentially involving competition for lagging strand binding, directs accessory helicase function to the leading strand at blocked forks.
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