Even though Et modification of DNA delayed
Even though Et743 modification of DNA delayed the progress of the translocating enzyme significantly, it did not trap RecBCD on DNA, nor did it alter the activity of the enzyme significantly. Under catalytic conditions, no loss of enzyme activity was observed (Table 2), nor were stable unwinding intermediates observed in agarose gels (Figure 5, Figure 7). To further verify that the enzyme was not being altered permanently, unmodified dsDNA was added to unwinding reactions using Et743-modifed DNA that had been allowed to proceed to completion. This second aliquot of DNA was unwound at a rate comparable to that of control reactions (data not shown). This indicates that the modification of DNA by Et743 affects the translocating RecBCD enzyme while it is in transit through the DNA. Enzyme trapping or inactivation by the drug was observed, however, at high concentrations of Et743 (288 drug Phos-tag per DNA molecule; data not shown). This drug to DNA molecular ratio corresponds to an average of one drug molecule every 14 bp, a value within the limits of the translocation step size of the enzyme of 23 nt. Presumably, at these high concentrations of Et743, each time the enzyme attempts to translocate 23 bp on dsDNA, it could do so into a region of modification. Since the modification by Et743 disrupts the interaction of the leading domain of RecB with the DNA, this would prevent the enzyme from moving, resulting in trapping or sequestering of the enzyme. Even though RecBCD is able to unwind and cleave Et743-modified dsDNA, χ-recognition was completely inhibited. This occurs due to the sequence selectivity of Et743 (5′-A, 5′-C) which overlaps that of χ at both the 5′-end and the 3′-end (5′-TGGT-3′). The recombination hotspot χ is recognized as the unwound single strand of DNA by the RecC subunit as the 3′-terminated DNA strand passes through the channel in RecC., The addition of the bulky adduct to χ impairs the ability of the RecC subunit to read the sequence, thereby preventing the recognition and response to χ from taking place. Modification of the χ sequence impairing the ability of RecBCD to recognize χ is not without precedence. A similar impairment has been observed with alternate χ sites, mutated at the 5′-G of the octanucleotide sequence, resulting in a reduction in χ-dependent recombination proficiency to 6% relative to that of wild-type χ. Thus, the proposed modification of χ by Et743 results in a similar inability of RecBCD to recognize the unwound, single-stranded χ-sequence, resulting in elimination of χ-fragment production. The ability of Et743 to affect accurate sequence reading of unwound DNA by RecBCD may be applicable to enzymes such as RNA polymerase, which reads the sequence of the template strand during transcription. Here, the presence of bulky adducts could both delay the progress of the advancing transcription machinery, and alter its ability to read the sequence accurately and to transcribe the DNA accurately. Adozelesin was able to inhibit RecBCD but clearly was not as effective as Et743. The result of adozelesin-induced modification of dsDNA is to bend, stiffen and stabilize the duplex by 20 deg.C. The combination of these effects results in biphasic time-courses in both helicase assays, and the production of discrete-sized intermediates and apparent χ-specific fragments in agarose gels. These effects are linked and can be explained by two models. In the first model, modification by adozelesin results in trapping of RecBCD during translocation and DNA unwinding. In the fluorimetric assay, using stoichiometric amounts of enzyme and 99 adozelesin molecules per DNA molecule, a brief (3 s) initial phase of unwinding was observed, followed by a second, longer phase in which a slower rate of unwinding was observed (18(±8) bp s−1). The biphasic time-course suggests that RecBCD is able to unwind a small segment of the duplex (approximately 363 bp) with a reduced rate (61(±28) bp s−1) relative to that observed on unmodified dsDNA (105(±27) bp s−1). During this period, eight to nine adozelesin molecules or, on average, one every 42 bp are encountered, resulting in the progress of the enzyme being impeded. In addition, and during this period, some fraction of the DNA-bound enzyme population becomes trapped or sequestered on the DNA. This results in the formation of unwinding intermediates that are visualized readily in agarose gels (Figures 5(a) and 8).