DNA stretching by bacterial initiators promotes replication origin opening

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Replication initiators are crucial for DNA replication, often forming oligomers to process origins.
• Cellular initiators contain AAA+ (ATPases associated with various cellular activities) domains for protein assembly and origin recognition.
• In bacteria, DnaA's AAA+ domain is hypothesized to aid in single-stranded DNA formation during origin melting.

Purpose of the Study:

• To elucidate the mechanism by which Aquifex aeolicus DnaA opens bacterial replication origins.
• To investigate the structural basis of DnaA's interaction with single-stranded DNA.
• To compare DnaA's DNA-binding mechanism with other AAA+ systems.

Main Methods:

• X-ray crystallography to determine the structure of DnaA oligomers.
• Solution-based biophysical techniques to study protein-DNA interactions.
• Strand-displacement assays to assess DNA unwinding activity.

Main Results:

• ATP-dependent assembly of Aquifex aeolicus DnaA into a spiral oligomer.
• The oligomer presents a continuous surface for successive AAA+ domains to bind and extend single-stranded DNA.
• DnaA binding induces a DNA conformation that prevents complementary strand annealing, distinct from RecA.
• DnaA utilizes an ATP-dependent stretching mechanism for origin opening.

Conclusions:

• DnaA directly opens replication origins through an ATP-dependent DNA stretching mechanism.
• The spiral oligomer formation is key to DnaA's origin-melting function.
• DnaA's DNA engagement mechanism shares similarities with other nucleic-acid-dependent AAA+ systems.