Archaeal Orc1 protein interacts with T-rich single-stranded DNA

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA replication initiation proteins form nucleoprotein complexes at replication origins.
• Prokaryotic and eukaryotic initiators interact with single-stranded DNA (ssDNA), a process essential for replication.
• The ssDNA interaction of archaeal replication initiators remained uncharacterized.

Purpose of the Study:

• To investigate whether archaeal DNA replication initiators interact with ssDNA.
• To characterize the ssDNA binding properties of the Aeropyrum pernix Orc1 (ApOrc1) protein.

Main Methods:

• Purified ApOrc1 protein was used.
• Binding assays included Bio-layer interferometry, surface plasmon resonance, and microscale thermophoresis.
• Interaction with ssDNA containing the Ori1 AT-rich region, polyT, and polyA was analyzed.

Main Results:

• ApOrc1 demonstrated the ability to interact with ssDNA.
• The protein showed preferential binding to thymidine-rich ssDNA.
• ATP hydrolysis was not necessary for ApOrc1-ssDNA interaction.

Conclusions:

• Archaeal replication initiator ApOrc1 interacts with ssDNA.
• This interaction is sequence-specific, favoring T-rich regions.
• ssDNA binding by ApOrc1 is independent of ATP hydrolysis, suggesting a distinct role in replication initiation.