Peptide inhibitors of DNA cleavage by tyrosine recombinases and topoisomerases.
The study of biochemical pathways requires the isolation and characterization of each and every intermediate in the pathway. For the site-specific recombination reactions catalyzed by the bacteriophage lambda tyrosine recombinase integrase (Int), this has been difficult because of the high level of efficiency of the reaction, the highly reversible nature of certain reaction steps, and the lack of requirements for high-energy cofactors or metals. By screening synthetic peptide combinatorial libraries, we have identified two related hexapeptides, KWWCRW and KWWWRW, that block the strand-cleavage activity of Int but not the assembly of higher-order intermediates. Although the peptides bind DNA, their inhibitory activity appears to be more specifically targeted to the Int-substrate complex, insofar as inhibition is resistant to high levels of non-specific competitor DNA and the peptides have higher levels of affinity for the Int-DNA substrate complex than for DNA alone. The peptides inhibit the four pathways of Int-mediated recombination with different potencies, suggesting that the interactions of the Int enzyme with its DNA substrates differs among pathways. The KWWCRW and KWWWRW peptides also inhibit vaccinia virus topoisomerase, a type IB enzyme, which is mechanistically and structurally related to Int. The peptides differentially affect the forward and reverse DNA transesterification steps of the vaccinia topoisomerase. They block formation of the covalent vaccinia topoisomerase-DNA intermediate, but have no apparent effect on DNA religation by preformed covalent complexes. The peptides also inhibit Escherichia coli topoisomerase I, a type IA enzyme. Finally, the peptides inhibit the bacteriophage T4 type II topoisomerase and several restriction enzymes with 2000-fold lower potency than they inhibit integrase in the bent-L pathway.
Klemm
M
M
Department of Biology and Molecular Biology Institute, San Diego State University, San Diego, CA, 92182, USA.
Cheng
C
C
Cassell
G
G
Shuman
S
S
Segall
A M
AM
D000595:Amino Acid Sequence; D001287:Attachment Sites, Microbiological / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism; D017122:Bacteriophage T4 / Q000201:enzymology; D010582:Bacteriophage lambda / Q000201:enzymology; D001483:Base Sequence; D002384:Catalysis / Q000187:drug effects; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D020128:Inhibitory Concentration 50; D019429:Integrase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D019426:Integrases / Q000378:metabolism*; D034982:Integration Host Factors; D007700:Kinetics; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009994:Osmolar Concentration; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D011995:Recombination, Genetic / Q000187:drug effects / Q000235:genetics; D013379:Substrate Specificity; D059004:Topoisomerase I Inhibitors; D014615:Vaccinia / Q000201:enzymology
D001426:Bacterial Proteins; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D019429:Integrase Inhibitors; D034982:Integration Host Factors; D010455:Peptides; D059004:Topoisomerase I Inhibitors; D004247:DNA; D019426:Integrases; D004262:DNA Restriction Enzymes; D004264:DNA Topoisomerases, Type I