Atomic force microscopy of DNA in solution and DNA modelling show that structural properties specify the eukaryotic...

Area of Science:

• Molecular Biology
• Genetics
• Biophysics

Background:

• Eukaryotic replication origins (ORIs) are A+T-rich, lack consensus sequences, but involve conserved proteins.
• DNA structure is hypothesized to be crucial for ORI function despite sequence variability.

Purpose of the Study:

• To investigate the structural properties of the Spars1 replication origin in Schizosaccharomyces pombe.
• To determine how DNA structure influences protein binding and initiation site selection.

Main Methods:

• Atomic force microscopy (AFM) in solution.
• DNA modeling and structural analysis.

Main Results:

• The Spars1 origin is structurally less stable and contains intrinsically bent, supercoiled DNA elements.
• The pORC-binding site overlaps with a superhelical region, and the replication initiation site (RIP) is within a curved DNA region.
• pORC unwinding shifts the RIP towards the apex of DNA curvature, promoting melting.

Conclusions:

• DNA structural dynamics, including bending and supercoiling, are critical for eukaryotic ORI function and initiation.
• The identified structural features in Spars1 are conserved in other eukaryotic origins, supporting a universal mechanism.