Sin resolvase catalytic activity and oligomerization state are tightly coupled.
Serine recombinases promote specific DNA rearrangements by a cut-and-paste mechanism that involves cleavage of all four DNA strands at two sites recognized by the enzyme. Dissecting the order and timing of these cleavage events and the steps leading up to them is difficult because the cleavage reaction is readily reversible. Here, we describe assays using activated Sin mutants and a DNA substrate with a 3'-bridging phosphorothiolate modification that renders Sin-mediated DNA cleavage irreversible. We find that activating Sin mutations promote DNA cleavage rather than simply stabilize the cleavage product. Cleavage events at the scissile phosphates on complementary strands of the duplex are tightly coupled, and the overall DNA cleavage rate is strongly dependent on Sin concentration. When combined with analytical ultracentrifugation data, these results suggest that Sin catalytic activity and oligomerization state are tightly linked, and that activating mutations promote formation of a cleavage-competent oligomeric state that is normally formed only transiently within the full synaptic complex.
Mouw
Kent W
KW
Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
Steiner
Andrew M
AM
Ghirlando
Rodolfo
R
Li
Nan-Sheng
NS
Rowland
Sally-J
SJ
Boocock
Martin R
MR
Stark
W Marshall
WM
Piccirilli
Joseph A
JA
Rice
Phoebe A
PA
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011995:Recombination, Genetic; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D050505:Mutant Proteins; D009841:Oligonucleotides; D004247:DNA; D004254:DNA Nucleotidyltransferases; C086893:sin protein, Staphylococcus aureus