It is conceivable however that the distortion
It is conceivable, however, that the distortion induced by adozelesin in dsDNA may decrease the processivity of the translocating RecBCD enzyme so that it dissociates more frequently from adducted DNA relative to unmodified DNA. Premature termination from the DNA would also produce partially unwound intermediates. Although this may be occurring it does not explain the biphasic time-courses. The loss of RecBCD activity during subsequent rounds of DNA unwinding under catalytic conditions is consistent with transient, adozelesin-induced enzyme trapping on the DNA (Table 2). The delayed appearance of ssDNA under stoichiometric conditions at later times in reactions is also consistent with enzyme entrapment. Therefore, we attribute the formation of stable intermediates visualized in agarose gels to trapping of the translocating RecBCD enzyme (although we cannot exclude pseudo-χ recognition entirely), as observed for other DNA helicases. Hedamycin was the least effective agent used in this study. Although this agent intercalates into DNA in the major groove, it does interact with the minor groove but to a lesser extent. Both hedamycin and Et743 alter the structure of the duplex significantly, protrude partially from DNA and increase the melting temperature of the duplex. Et743 interacts exclusively with the minor groove and is the more potent inhibitor, suggesting that modification of the minor groove is more effective in disrupting RecBCD enzyme-catalyzed DNA unwinding than is intercalation between base-pairs. Although binding of hedamycin produces a 20 deg. C increase in the DNA melting temperature, modification of dsDNA by hedamycin did not inhibit the progress of RecBCD significantly, as only a modest reduction in unwinding rate and little to no effect on extent was observed. Significant inhibition did occur, however, at elevated drug concentrations where, on average, one drug molecule would be encountered every 5 bp (Figure 7). Similar results for intercalating dye idasanutlin (e.g. ethidium bromide) on RecBCD have been observed using fluorescence-based assays. As for hedamycin, little or no inhibition is observed at low concentrations of ethidium bromide, while significant inhibition requires elevated concentrations of dye. The effects of hedamycin on the unwinding of DNA by RecBCD are complicated by the presence of free, unreacted drug molecules. It was the only agent used here where unbound drug molecules affected enzyme activity, as a greater level of inhibition of the rate of unwinding was observed using non-precipitated DNA, which contained detectable levels of free drug molecules (Table 1). Even though free adozelesin was present following treatment with the drug, as shown in the heat-induced, DNA strand cleavage assay, free adozelesin did not affect either SSB or RecBCD (data not shown; and see Table 1). Incubation of RecBCD with hedamycin in the absence of DNA resulted in slower rates of unwinding (e.g. at 30 μM a 50% decrease compared to the control was observed, data not shown). In addition, free hedamycin inactivated SSB as extended incubations of drug and protein resulted in decreased ability of SSB to bind to unmodified ssDNA (data not shown). Consequently, experiments using hedamycin were done using drug-treated and ethanol-precipitated dsDNA. The rate of unwinding of ethanol-precipitated dsDNA was reduced by 35% relative to the control, indicating that DNA-bound hedamycin is able to slow the progress of the translocating RecBCD enzyme, albeit not very effectively. The intercalation of hedamycin into dsDNA resulted in a novel and unanticipated effect on the nuclease activity and χ-recognition of RecBCD. Unlike adozelesin or Et743, hedamycin did not completely prevent the recognition and response of RecBCD to χ. Instead, formation of the downstream, top-strand χ-specific fragment still occurred, albeit at a level reduced by twofold compared to the control reaction (Figure 8, top-strand, (ii),). As both the levels of full-length ssDNA and that of the bottom-strand, upstream χ-specific fragment were reduced significantly, this suggests that the bottom-strand nuclease activity was activated constitutively. A gating mechanism has been proposed to control the access of each unwound single strand of DNA to the single nuclease site in RecB. Thus, the alkylation of the DNA by hedamycin perturbs the gating mechanism so that the 5′-terminated strand relative to the entry point of the enzyme (the bottom strand on which RecD translocates) is exposed more frequently to the nuclease domain. Consequently, before interacting with χ, both strands of DNA are cleaved frequently. As the ability of the enzyme to recognize and respond to χ is largely unaffected, nuclease activity of the top strand is still attenuated, resulting in the production of the downstream, top-strand χ-specific fragment. Formation of the bottom-strand, upstream χ-specific fragment does not occur, since it was prematurely degraded.