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  • To understand more clearly how


    To understand more clearly how these activities are combined in the heterotrimeric enzyme and to understand more clearly how DNA alkylation might effect DNA helicase activity, we summarize the relevant features of the structure of RecBCD determined in the presence of DNA. The RecB and RecC subunits are intimately associated with one another, Q-VD(OMe)-OPh with the RecD subunit being more peripheral (Figure 2(a)). The RecB and RecD subunits couple the hydrolysis of ATP to DNA translocation and strand separation by pulling DNA into the holoenzyme, and through the RecC subunit, where strand separation occurs., Although RecC has no demonstrable enzymatic activity, it acts as a scaffold onto which the two motors assemble, it is intimately involved in strand separation and is responsible for χ recognition., Finally, the enzyme contains a single nuclease active site that resides within the C terminus of the RecB subunit, and which is positioned approximately opposite the entry point of dsDNA into the holoenzyme. Unwinding of dsDNA by RecBCD involves an intimate coordination between subunits, and is proposed to occur in several sequential steps, utilizing a quantum inchworm mechanism. First, the enzyme binds to dsDNA ends with the leading domain of RecB reaching out 23 nt ahead of the ssDNA/dsDNA junction. Here, it interacts with the minor groove of the duplex and initially anchors the enzyme in place (Figure 2)., Thereafter, this domain translocates the duplex into the enzyme, where it is separated into ssDNA by RecC (Figure 2(b)). The unwound single strands of DNA pass through 11–16 Å diameter Q-VD(OMe)-OPh in RecC (one each to RecB and RecD; Figure 2(b), inset). Each motor translocates on the unwound single strands of DNA and, as a consequence, the unwound ssDNA is pulled through RecC to the nuclease active site, where it is subsequently cleaved. To ascertain how bulky adducts resulting from intra-strand alkylation might perturb the function of RecBCD, we selected three distinct DNA alkylation agents. The three agents, adozelesin, ecteinascidin 743 (Et743) and hedamycin, possess unique structures and sequence selectivity; they covalently attach bulky adducts and significantly alter the structure of DNA (Figure 3). These molecules bind to dsDNA and alkylate only one strand of the duplex in cis, and have been shown to inhibit DNA replication,32., 33., 34. and repair., Below, we present the relevant details of each these agents. Adozelesin belongs to the family of extremely cytotoxic, cyclopropylpyrroloindole anticancer agents that irreversibly and covalently associate with DNA in the minor groove. Adozelesin consists of three subunits, A (a cyclopropyl unit), B (an indole unit), and C (benzofuran) (Figure 3(a)). It binds to DNA in the minor groove at PuPy/PuTTA sequences, and modifies the 3′ adenine via its cyclopropyl moiety. The interaction of CC-1065 with DNA, adozelesin\'s parent agent, has provided a model for the structural features of the adozelesin–DNA complex. Both CC-1065 and adozelesin bend and stiffen the double helix, increasing the melting temperature of the DNA by as much as 20 deg. C. On binding to DNA, CC-1065 widens the minor groove, due to the ethano bridges between the B and C subunits. As adozelesin has an amide linker at the same position, it is unable to widen the DNA minor groove to the same extent as CC-1065., Instead, adozelesin forms strong hydrogen bonds between the amide linker of the indole and benzofuran subunits, and the carbonyl group of the central thymine. Consequently, this positions the drug not centrally within the minor groove, but instead towards the modified DNA strand. As a consequence of DNA duplex distortion and alkylation, both adozelesin and CC-1065 have been shown to inhibit DNA ligase, DNA polymerase and DNA helicases in vitro.42., 43., 44. Ecteinascidin 743 (Et743) is an anticancer antibiotic isolated from the Caribbean tunicate Ecteinascidia turbinate. It is a carbinolamine-containing, DNA-binding agent consisting of three fused tetrahydroisoquinoline rings, the A, B and C subunits (Figure 3(b)). The A and B subunits are responsible for DNA sequence recognition and bonding, while the C subunit, which does not contact the DNA, protrudes out of the minor groove perpendicular to the duplex. The C subunit is not inert, it plays an important role in the cytotoxicity of Et743. It targets DNA by binding in the minor groove in G+C-rich sequences, alkylates the amino group of guanine residues at position 2, and bends DNA toward the major groove with an absolute curvature of 17(±3)°. The direction of bending is a novel feature among minor groove DNA-interactive agents, making Et743 unique. Binding to the minor groove occurs through an extensive network of hydrogen bonds that presumably results in an increase in duplex stabilization by 13–19 deg. C. Following binding, alkylation of predominantly guanine bases, located either in the sequence 5′-PuGC or 5′-PyGC, occurs., Other adducts are formed, but these are less stable and are reversible., Et743 is active against soft tissue sarcomas, in breast and ovarian cancers and is currently in phase II/III clinical trials., It effectively disrupts transcription factor complexes in vitro and inhibits gene expression in vivo., Et743 DNA adducts also inhibit nucleotide excision repair enzymes both in vitro and in vivo during the excision step, causing the accumulation of DNA–drug–protein intermediates.,