The RecG branch migration protein of Escherichia coli dissociates R-loops

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• RuvAB and RecG are essential helicases in Escherichia coli, facilitating Holliday junction branch migration during genetic recombination.
• Both proteins unwind and rewind DNA at the junction point, playing critical roles in DNA repair and replication processes.

Purpose of the Study:

• To investigate the helicase activities of RuvAB and RecG using RNA:DNA hybrid molecules.
• To compare the substrate specificities and functional differences between RuvAB and RecG in DNA unwinding and R-loop processing.

Main Methods:

• Utilized RNA:DNA hybrid molecules of varying lengths (218 bp and 35 bp) to assess helicase activity.
• Examined the ability of RuvAB and RecG to unwind partial DNA duplexes and remove RNA from R-loop structures.
• Assessed the effect of RecG on plasmid copy number in E. coli.

Main Results:

• RuvAB unwound RNA:DNA partial duplexes of at least 218 bp, requiring RuvA, RuvB, ATP, and Mg2+.
• RecG did not unwind these RNA:DNA duplexes, even with a reduced duplex region of 35 bp.
• RecG effectively removed a 218 nt RNA from an R-loop substrate, a function not performed by RuvAB.
• RecG expression significantly reduced the copy number of ColE1-based plasmids, suggesting interference with replication initiation.

Conclusions:

• RecG possesses distinct R-loop dissociation activity, differentiating it from RuvAB's DNA unwinding function.
• RecG's ability to resolve R-loops likely contributes to its role in regulating plasmid replication and potentially in homologous recombination.
• The findings provide insights into the specialized functions of RecG and RuvAB in DNA metabolism, including recombination and replication priming.