Identification of strong DNA binding motifs for bleomycin

The bleomycins (BLMs) are clinically used antitumor antibiotics. Their mechanism of action is believed to involve oxidative cleavage of DNA and possibly also RNA degradation. DNA degradation has been studied extensively and shown to involve binding of an activated metallobleomycin to DNA, followed by abstraction of C4'-H from deoxyribose in the rate-limiting step for DNA degradation. It is interesting that while DNA and RNA degradation by activated Fe center dot BLM has been studied extensively, much less is known about the actual binding selectivity of BLM, that is, the obligatory step that precedes cleavage. Thus it is unclear whether cleavage specificity is defined by the binding event or whether cleavage occurs at a subset of preferred binding sites. With only a few exceptions, NMR binding studies have employed metalloBLMs such as Co center dot BLM and Zn center dot BLM whose therapeutic relevance is uncertain. A single biochemical study that compared DNA binding and cleavage directly also employed Co center dot BLM. It is logical to anticipate that preferred sites of DNA cleavage will occur at sites that are (a subset of) preferred DNA binding sites, but there are currently no data available relevant to this issue. Herein, we describe the development and implementation of a novel strategy to identify DNA motifs that bind BLM strongly.