Inhibition of RecBCD enzyme by antineoplastic DNA alkylating agents.
To understand how bulky adducts might perturb DNA helicase function, three distinct DNA-binding agents were used to determine the effects of DNA alkylation on a DNA helicase. Adozelesin, ecteinascidin 743 (Et743) and hedamycin each possess unique structures and sequence selectivity. They bind to double-stranded DNA and alkylate one strand of the duplex in cis, adding adducts that alter the structure of DNA significantly. The results show that Et743 was the most potent inhibitor of DNA unwinding, followed by adozelesin and hedamycin. Et743 significantly inhibited unwinding, enhanced degradation of DNA, and completely eliminated the ability of the translocating RecBCD enzyme to recognize and respond to the recombination hotspot chi. Unwinding of adozelesin-modified DNA was accompanied by the appearance of unwinding intermediates, consistent with enzyme entrapment or stalling. Further, adozelesin also induced "apparent" chi fragment formation. The combination of enzyme sequestering and pseudo-chi modification of RecBCD, results in biphasic time-courses of DNA unwinding. Hedamycin also reduced RecBCD activity, albeit at increased concentrations of drug relative to either adozelesin or Et743. Remarkably, the hedamycin modification resulted in constitutive activation of the bottom-strand nuclease activity of the enzyme, while leaving the ability of the translocating enzyme to recognize and respond to chi largely intact. Finally, the results show that DNA alkylation does not significantly perturb the allosteric interaction that activates the enzyme for ATP hydrolysis, as the efficiency of ATP utilization for DNA unwinding is affected only marginally. These results taken together present a unique response of RecBCD enzyme to bulky DNA adducts. We correlate these effects with the recently determined crystal structure of the RecBCD holoenzyme bound to DNA.
Dziegielewska
Barbara
B
Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Beerman
Terry A
TA
Bianco
Piero R
PR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000495:Allosteric Site / Q000187:drug effects; D000880:Anthraquinones / Q000737:chemistry / Q000494:pharmacology; D018906:Antineoplastic Agents, Alkylating / Q000737:chemistry / Q000494:pharmacology*; D001572:Benzofurans; D002384:Catalysis / Q000187:drug effects; D003509:Cyclohexanecarboxylic Acids / Q000737:chemistry / Q000494:pharmacology; D053138:Cyclohexenes; D004247:DNA / Q000737:chemistry*; D004265:DNA Helicases / Q000037:antagonists & inhibitors; D000080890:Duocarmycins; D004926:Escherichia coli / Q000201:enzymology*; D043211:Exodeoxyribonuclease V / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005453:Fluorescence; D006868:Hydrolysis / Q000187:drug effects; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology; D008958:Models, Molecular; D009690:Nucleic Acid Conformation / Q000187:drug effects; D011995:Recombination, Genetic / Q000187:drug effects
D000880:Anthraquinones; D018906:Antineoplastic Agents, Alkylating; D001572:Benzofurans; D003509:Cyclohexanecarboxylic Acids; D053138:Cyclohexenes; D000080890:Duocarmycins; D007211:Indoles; C001328:hedamycin; C060090:adozelesin; D000255:Adenosine Triphosphate; D004247:DNA; D043211:Exodeoxyribonuclease V; D004265:DNA Helicases