Figure 4.

Initial mapping of cross-link sites by RNase H cleavage. (A) Mapping of cross-links generated with the primer S-5 6-mer. The RNase H cleavage products of RNA–DNA hybrids (thin arrows) were generated by the procedure described in Figure 3A. The bold arrow denotes uncleaved primer DNA–RNA molecules. Lane 1: no RNase H, no complementary oligo. Lane 2: RNase H cleavage products of a hybrid prepared with an oligo complementary to a region of TER spanned by the nucleotides 57–72. Lane 3: RNase H cleavage products of a hybrid formed with an oligo complementary to a region of TER spanned by the nucleotides 41–60. Lane 4: RNase H cleavage products of a hybrid formed with an oligo complementary to a region of TER spanned by the nucleotides 24–40. Lane 5: no complementary oligo. The size markers on the left are φX174 DNA/HinfI fragments (Promega) that were radioactively labeled with [γ-³²P] ATP, using T4 polynucleotide kinase. (B) Same RNase H cleavage assays as in A, except that RNA–DNA hybrids were formed with uniformly labeled TER synthesized in vitro, as described in ‘Materials and Methods’ section. These mixtures were not UV irradiated. (C) Schematic drawing of the RNase H cleavage products shown in A and of cleavage products obtained in similar assays performed with other indicated complementary oligos. The solid and the dashed two headed arrows denote the radioactively labeled and the unlabeled fragments, respectively. The dashed vertical lines denote the region including the cross-link. (D) Mapping of cross-link generated with the primer S-20 21-mer. Lane 1: no RNase H, no complementary oligo. Lane 2: RNase H cleavage products of a hybrid prepared with an oligo complementary to a region of TER spanned by the nucleotides 35–49. Lane 3: RNase H cleavage products of a hybrid formed with an oligo complementary to a region of TER spanned by the nucleotides 30–46. Lane 4: RNase H cleavage products of a hybrid formed with an oligo complementary to a region of TER spanned by the nucleotides 24–40. Lane 5: no complementary oligo. (E) Same RNase H cleavage assays as in D, except that RNA–DNA hybrids were formed with the uniformly labeled TER synthesized in vitro. (F) Schematic drawing of the RNase H cleavage products shown in D and of the cleavage products obtained in similar assays performed with other indicated complementary oligos. Notations are as in C.