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Binding and cleavage of nicked substrates by site-specific recombinases XerC and XerD
G W Blakely1, A O Davidson, D J Sherratt
1Microbiology Unit, Department of Biochemistry, University of Oxford, UK.
Abstract:
In Xer site-specific recombination two related recombinases, XerC and XerD, catalyse strand cleavage and rejoining reactions at a site, dif, in order to ensure normal chromosome segregation during cell division in Escherichia coli. We have used nicked suicide substrates to trap reaction intermediates and show that XerC cleaves the top strand efficiently while XerD is less efficient at cleaving the bottom strand of dif. Recombinase-mediated cleavage positions are separated by six base pairs and occur at either end of the dif central region adjacent to the recombinase binding sites. XerC can cleave the top strand of dif inefficiently in the absence of its partner recombinase during a reaction that does not require intermolecular synapsis. The presence of a nick in the bottom strand of dif allows cooperative interactions between two XerC protomers bound to adjacent binding sites, suggesting that a conserved interaction domain is present in both XerC and XerD. Cooperativity between two identical recombinase protomers does not occur on un-nicked linear DNA. Ethylation interference footprinting of two XerD catalytic mutant proteins suggests that the conserved domain II arginine from the integrase family RHRY tetrad may make direct contact with the scissile phosphate.
Insights
XerC and XerD recombinases are crucial for E. coli chromosome segregation. Studies reveal XerC efficiently cleaves the top DNA strand, while XerD is less efficient on the bottom strand at the dif site.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Xer site-specific recombination is essential for proper chromosome segregation in Escherichia coli.
- The recombinases XerC and XerD mediate DNA cleavage and rejoining at the dif site.
Purpose of the Study:
- To investigate the cleavage efficiency and intermediate trapping of XerC and XerD at the dif site.
- To elucidate the mechanism of strand cleavage and the role of DNA nicks in Xer recombination.
Main Methods:
- Utilized nicked suicide substrates to trap reaction intermediates.
- Employed ethylation interference footprinting with XerD catalytic mutant proteins.
Main Results:
- XerC demonstrates efficient cleavage of the top strand of dif, whereas XerD is less efficient on the bottom strand.
- Cleavage sites are separated by six base pairs at the dif central region.
- DNA nicks facilitate cooperative interactions between XerC protomers, suggesting conserved interaction domains in XerC and XerD.
- Identified potential direct contact between conserved domain II arginine and the scissile phosphate in XerD.
Conclusions:
- The study clarifies the distinct roles of XerC and XerD in dif site cleavage.
- DNA nicks are critical for cooperative recombinase binding and activity.
- A conserved interaction domain likely exists between XerC and XerD, with potential involvement of arginine in catalysis.