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DNA topoisomerase I (Top1) acts as a swivel to alleviate DNA overwinding in advance of replication forks and DNA supercoiling produced by RNA polymerases tracking along the DNA. Top1 cleaves one strand of duplex DNA and becomes covalently linked to the 3′ DNA end by a phosphotyrosyl bond. The nicked strand rotates around the uncleaved strand to relax supercoiled DNA. A second transesterification religates the DNA. Top1 is also the cellular target of camptothecin (CPT), analogs of which have significant activity against human cancers. CPT reversibly stabilizes the covalent Top1-DNA intermediate. Yet, we still lack sufficient insight into the consequences of Top1 poisoning to explain the S-phase dependence of these drugs. We exploited the yeast system to define mutations in Top1 that alter enzyme sensitivity to CPT. Top1 forms a protein clamp around duplex DNA, where opposable Lip domains form a salt bridge to complete the protein ring. Our analyses of active site mutations suggest that the unique architecture of Top1, where a flexible pair of extended α-helices (linker domain) position the C-terminal active site tyrosine domain within the catalytic pocket of the enzyme clamp, is an important determinant of cell sensitivity to CPT. In structures of human Top1-DNA covalent complexes, a conserved Gly residue lies at one end of an α-helix that contains the active site tyrosine linked to DNA. This is preceded by Leu, which connects the flexible linker with the active site domain. When drug is bound, the linker becomes rigid and is oriented at an oblique angle from the DNA helix. This is accompanied by an extension of the α-helix to include Gly and Leu. The orientation of Leu is also rotated such that it now interacts with the C-terminus of linker α-helix. We report that mutation of this conserved Gly has profound effects on yeast and human Top1 sensitivity to CPT. Moreover, the spectrum of CPT sensitivity of the mutant yeast enzymes in vitro mirrored that observed in yeast cells. Mutating Gly to polar residues Gln or Asn increased Top1 sensitivity to CPT. This was further enhanced by acidic residues Asp or Glu, which also induced a 20-40 fold increase in cell sensitivity to CPT, relative to that observed with wild-type Top1. The catalytic activity of the mutant proteins was similar to wild-type Top1, yet, mutation-induced alterations in active site architecture were revealed by tryptic digests of DNA cleavage/religation assays. In Ramachandron plots, substitutions that increased yeast Top1 sensitivity to CPT are predicted to stabilize this α-helix. By contrast Val, which renders Top1 resistant to CPT, is predicted to destabilize an α-helix. As alterations in linker flexibility have been posited to affect Top1 sensitivity to CPT, our findings suggest that the conserved Gly provides the flexible hinge that enables linker movement, while the preceding Leu constrains linker movement to facilitate CPT binding.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA