Three-stranded paranemic joints: architecture, topological constraints and movement

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Paranemic joining is a DNA strand exchange reaction catalyzed by RecA and SSB proteins.
• This process joins homologous DNA molecules that lack homologous ends, a crucial step in DNA repair and recombination.
• Understanding the structural intermediates of paranemic joining is essential for elucidating its mechanism.

Purpose of the Study:

• To investigate the structural characteristics of joint complexes formed during paranemic joining.
• To visualize the interaction between RecA-bound single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) using electron microscopy.
• To determine the role of SSB protein in the formation and structure of these joint complexes.

Main Methods:

• Electron microscopy (EM) was employed to examine DNA joint complexes.
• A novel fast-freezing and freeze-drying technique was used for sample preparation, avoiding chemical fixation.
• EM immunogold staining and psoralen photo-crosslinking were utilized to identify protein localization and DNA proximity.

Main Results:

• Long joint complexes (several kilobases) were observed between RecA-coated ssDNA filaments and linear dsDNA.
• The presynaptic filament structure was disorganized in the presence of dsDNA, with SSB protein localized to these regions.
• Psoralen crosslinking indicated that only a short region (200-300 bp) involved close proximity of all three DNA strands.

Conclusions:

• RecA and SSB proteins can mediate the formation of extensive three-stranded DNA structures during paranemic joining.
• The observed structural disorganization suggests a dynamic process involving DNA strand rearrangement.
• A model is proposed where a short paranemic joint moves along the RecA-presynaptic filament to facilitate extensive joining.