br Author Contributions br Acknowledgments br
Introduction Many anticancer agents bind covalently to DNA, introduce bulky adducts, and inhibit DNA metabolic processes including repair, replication and transcription.1., 2., 3. Frequently, the first enzymes to encounter DNA adducts are DNA helicases. These enzymes progress in a unidirectional manner through the DNA helix and unwind the duplex producing nascent single-stranded DNA (ssDNA) in a reaction fueled by energy derived from the hydrolysis of nucleoside 5′-triphosphates.4., 5., 6., 7. Consequently, if a bulky adduct disrupts helicase progression, this might translate into drug-induced inhibition of the repair, replication or transcription processes. Several DNA-binding drugs, including minor groove binders and intercalating agents, have been evaluated for their ability to inhibit DNA helicases in vitro. Several DNA helicases have been utilized as model systems, including mammalian helicase II, the Bloom\'s and Werner\'s proteins, simian virus 40 (SV40) large T-antigen (TAg), herpes simplex virus (HSV) UL9 and the Escherichia coli Rep, UvrD and RecBCD enzymes. These studies demonstrated that the level of drug-induced inhibition of DNA unwinding depended on both the agent used and on the DNA helicase being tested. For example, daunorubicin was shown to potently inhibit DNA unwinding by Tag and mammalian helicase II, but affected DNA unwinding by the bacterial UvrD and Rep helicases only moderately.9., 10., 11., 12. Similarly, intercalating agents inhibit UvrD, whereas minor groove-binding agents do not. In contrast, whereas minor groove-binding compounds are potent inhibitors of both the Bloom\'s and Werner\'s DNA helicases, intercalating agents do not inhibit these DNA helicases significantly. Thus, the ability of a DNA-binding Ranitidine to inhibit a DNA helicase is a combination of the properties of the agent being tested and those of the DNA helicase(s) being studied. Although the agents alluded to above and those used in this study exert their effects in eukaryotic cells, the well-characterized RecBCD enzyme of Escherichia coli is an excellent model enzyme to study the effects of DNA-damaging agents on DNA unwinding. This is the best characterized DNA helicase to date, with a large body of both in vivo and in vitro data available. In contrast to other DNA helicases, the RecBCD enzyme is the complete nanomachine, requiring no additional proteins to mediate efficient DNA unwinding. Second, although the enzyme unwinds double-stranded DNA (dsDNA) non-specifically, it does read the sequence of the unwound DNA as it passes through the core of the enzyme. Thus the ability of an agent to inhibit sequence-specific regulation can also be evaluated using this enzyme. Third, in addition to being well characterized both in vivo and in vitro, the crystal structure of the enzyme in the presence of DNA was determined recently. Thus, it is possible to correlate the in vitro effects of DNA adducts on DNA unwinding with the three-dimensional structure of the enzyme. RecBCD is a combination helicase/nuclease that is critical to homologous genetic recombination and DNA repair in E. coli., The holoenzyme consists of the three subunits RecB, RecC, and RecD. The RecB and RecD subunits are NTP hydrolysis-dependent DNA motor proteins that drive DNA translocation and strand separation,, while RecC functions in holoenzyme scaffolding, DNA unwinding and recognition of the recombination hotspot χ. Together, these subunits form a complex enzyme possessing DNA helicase, ATPase and nuclease activities. In vitro, RecBCD binds to blunt or almost blunt dsDNA, ends with >106-fold affinity relative to internal sites. The helicase unwinds the DNA at rates of up to ∼1000 bp s−1 at 37 °C, traversing on average 30,000 bp per binding event., DNA unwinding is accompanied by the simultaneous and asymmetric endonucleolytic degradation of unwound ssDNA with the 3′-terminated strand, relative to the entry point of the enzyme, being preferentially degraded (Figure 1(a)). For both translocation and DNA degradation to occur, RecBCD requires Mg2+ and the energy derived from the hydrolysis of a nucleoside triphosphate, typically ATP. The nuclease activity of RecBCD is regulated by the recombination hotspot χ (chi for crossover hotspot instigator; defined as 5′-GCTGGTGG-3′). The translocating RecBCD enzyme recognizes χ as the unwound single strand of DNA but only when approaching χ from the 3′-side (Figure 1(b)) Recognition of χ results in the translocating enzyme pausing at χ, during which the polarity of the nuclease activity is altered. The 3′ to 5′ activity is attenuated while the 5′ to 3′ nuclease activity is up-regulated. Consequently, continued translocation past χ results in RecBCD producing 3′-tailed ssDNA, onto which the RecA protein is preferentially loaded, thereby facilitating the initiation step of homologous recombination. In addition to controlling the polarity of the RecBCD nuclease activity, the encounter with χ results in an uncoupling of the two RecBCD helicase motors (RecB and RecD), so that DNA unwinding beyond χ is powered by the slower RecB helicase with the RecD motor having been inactivated at χ. Thus, a properly oriented χ-sequence controls the polarity of degradation and the rate of enzyme translocation.