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Holliday junction binding and resolution by the Rap structure-specific endonuclease of phage lambda
Gary J Sharples1, Fiona A Curtis, Peter McGlynn
1Centre for Infectious Diseases, Wolfson Research Institute, University of Durham, Queen's Campus, Stockton-on-Tees TS17 6BH, UK. gary.sharples@durham.ac.uk
Journal of Molecular Biology
|June 30, 2004
Summary
Rap endonuclease resolves complex DNA structures, including Holliday junctions with larger homologous cores, by nicking DNA. This function is crucial for phage lambda recombination and DNA repair pathways.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Rap endonuclease is known to nick DNA at branched structures.
- Previous studies showed Rap's limitations with specific Holliday junction substrates.
Purpose of the Study:
- To investigate Rap endonuclease's ability to resolve Holliday junctions with larger homologous cores.
- To understand Rap's mechanism and substrate specificity in DNA recombination.
Main Methods:
- Enzymatic assays using synthetic Holliday junction DNA substrates (50 bp and chi structures).
- Analysis of DNA cleavage products and their ligation by T4 DNA ligase.
- Assessment of Rap protein's effect on junction conformation in the presence/absence of magnesium ions.
Main Results:
- Rap successfully mediated symmetrical resolution of Holliday structures with larger homologous cores.
- Rap showed a weak preference for cleaving between 5'-GC dinucleotides on mobile junctions.
- Rap did not alter the global conformation of DNA junctions, suggesting a unique binding mode.
Conclusions:
- Rap endonuclease functions as a Holliday junction resolvase for specific substrates.
- Rap's non-conformational altering binding mode is distinct from other known junction-binding proteins.
- Rap plays a significant role in resolving branched DNA structures during phage recombination.