EFFICIENT FORMATION OF A CROSSLINKABLE HMT MONOADDUCT AT THE Kpn I RECOGNITION SITE |
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| Authors: | Howard Gamper Jacques Piette John E Hearst |
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| Institution: | Department of Chemistry, University of California, Berkeley, CA, and Melvin Calvin Laboratory, Division of Chemical Biodynamics, Lawrence Berkeley Laboratory, Berkeley, CA 94720, USA |
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| Abstract: | Abstract The double-stranded Kpn I linker, CGGTACCG, is readily crosslinked by 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT). Under identical conditions, the corresponding Bam HI linker, CGGATCCG, is resistant to modification. The differential reactivity of HMT towards the two sequences is also observed with SV40 DNA where the Kpn I restriction site is ten-fold more susceptible to HMT modification than the Bam HI site. Selective reaction with the Kpn I site is attributed to preferential intercalation of HMT into the TpA sequence. The availability of crosslinked Kpn I linker has allowed us to characterize the kinetics of photoreversal. Upon irradiation at 254 nm, the diadduct reverses at a faster rate than the monoadduct, leading to an accumulation of the latter. The resultant monoadduct reforms crosslink rapidly upon irradiation at 365 nm. DNA oligomers like the Kpn I linker, which can be modified by the above protocol to contain a crosslinkable HMT monoadduct, could he exploited as photoreactive sequence-specific probes. |
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