Resolution by unassisted Top3 points to template switch recombination intermediates during DNA replication.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The evolutionarily conserved Sgs1/Top3/Rmi1 (STR) complex is crucial for DNA replication and repair.
• A key function of the STR complex is dissolving toxic X-shaped recombination intermediates that form during replication of damaged DNA.
• The precise structure and resolution mechanisms of these X-shaped molecules remain under investigation.

Purpose of the Study:

• To investigate the role of Top3, independent of Sgs1 and Rmi1, in resolving DNA recombination intermediates.
• To elucidate the structural nature of X-shaped molecules accumulating during replication of damaged DNA.
• To clarify the specific substrates resolved by Top3 versus the entire STR complex.

Main Methods:

• Genetic approaches were employed to study DNA replication and repair.
• Two-dimensional gel electrophoresis of genomic DNA was used to analyze recombination intermediates.
• In vitro biochemical assays with purified Top3 were performed to assess its resolution activity on synthetic DNA structures.

Main Results:

• Top3, when acting alone, demonstrated a modest ability to confer resistance to methyl methanesulfonate (MMS) and resolve recombination-dependent X-shaped molecules.
• The analyzed X-shaped molecules exhibited structural characteristics consistent with hemicatenane-related template switch recombination intermediates (Rec-Xs), not Holliday junctions (HJs).
• Purified Top3 successfully resolved synthetic Rec-X structures in vitro but not synthetic double HJs, and did not influence crossing over during double-strand break repair.

Conclusions:

• Unassisted Top3 possesses distinct DNA resolution activities, primarily targeting Rec-X intermediates.
• These findings clarify that Top3's independent functions are specific to substrates different from HJs.
• The study illuminates the nature of X-shaped molecules during damaged DNA replication and refines our understanding of Top3 and STR complex roles in resolving replication-associated recombination intermediates.