New Approaches to DNA Mapping: Synthetic 

 Endonucleases 



Betsy M. Sutherland and Gary A. Epling 



Biology Department, Brookhaven National Laboratory. Upton. NY 1 1973 



(516)'282-3293. FTS 666-3293 



Recognition and mapping of functionally important DNA regions (e.g.. regulatory and 

 coding regions and initiation sequences) can he greatly facilitated by specific DNA 

 cleavage at such sites. Synthetic endonucleases. able to cleave at regions of functional 

 importance, will be created by coupling DNA site-specific binding proteins via linker 

 arms to light-activatable cleaving moieties: specific binding function is provided by the 

 DNA binding protein; cleavage activity is provided by the activatable cleaving 

 molecules. A prototype system of Rose-Bengal (RB) coupled via a hexanoic acid linker 

 to a DNA lesion site-specific monoclonal antibody will be developed for other specific 

 DNA-binding proteins, including the T7 RNA polymerase and mammalian transcription 

 initiation factors. 



Coupling of RB-hexanoic acid to T7 RNA polymerase via l-ethyl-3-(3- 

 dimethylaminopropyl) coupling (EDO was found to yield RB-tagged polymerase, 

 which can specifically bind to a plasmid containing a T7 promoter. However, the 

 conditions for EDC were sufficiently stringent to result in low final yields of active 

 polymerase. We designed and synthesized an RB triethylene glycol succinate activated 

 ester, which can be added to polymerase under buffer conditions optimal for enzyme 

 stability. Levels of the RB addition that yielded maximum specific binding of the 

 polymerase to T7 promoter sites were determined. Preliminary results indicate that the 

 RB-labeled T7 RNA polymerase can mediate the cleavage of T7 DNA to a level of at 

 least 2.4 cleavages per DNA molecule. The sites of cleavage by the tagged polymerase 

 are being determined. 



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