Rep protein accommodates together dsDNA and ssDNA which enables a loop-back mechanism to plasmid DNA replication...

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• DNA replication initiation in bacteria involves initiator proteins binding to double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) at the replication origin.
• The precise structural and functional mechanisms of these nucleoprotein complexes remain incompletely understood, with various models proposed.

Purpose of the Study:

• To elucidate the structural-functional relationship of plasmid replication initiator proteins (Rep) with DNA during replication initiation.
• To identify specific amino acid residues critical for the interaction between Rep proteins (TrfA, RepE) and ssDNA.

Main Methods:

• Crosslinking combined with mass spectrometry (MS) was employed to analyze protein-DNA interactions.
• Mutant protein analysis and the determination of crystal structures were utilized to understand complex formation.
• Crystal structures of RepE in complex with ssDNA and with both ssDNA and dsDNA were solved.

Main Results:

• Specific amino acid residues essential for RepE interaction with ssDNA were identified within the WH1 and WH2 domains.
• These ssDNA interaction sites are spatially distinct from the dsDNA recognition interface on RepE.
• The study demonstrated that Rep protein binding to dsDNA and interaction with ssDNA cannot occur in trans, highlighting their cooperative role in dsDNA melting at the DNA unwinding element (DUE).

Conclusions:

• The findings reveal a novel loop-back mechanism where a single plasmid replication initiator molecule simultaneously binds both dsDNA and ssDNA.
• This mechanism is essential for initiating DNA replication by facilitating dsDNA melting at the DUE.