Plasmid pTC27. The plasmid target sequence aligned with the ¹²⁵I-TFO, and restriction sites used to obtain smaller fragments for analysis. The 5-[¹²⁵I]-dC of the TFO is indicated in bold italics, and the “G” residue of the plasmid duplex targeted for Hoogsteen base pairing by the TFO is indicated in bold.

Enzymatic probing of the BglII fragment(s) 3′-end labeled upper strand. (A) The BglII fragment model for the longest sequence detected. An asterisk indicates the 3′-³²P label. The ¹²⁵I-TFO targeted duplex G (which is also the DSB breakage maximum) is indicated in lowercase bold type. The subscripted number permits correlation of this sequence to those in panels B-E and Fig. 1. (B-E) Densitometric quantification of sequencing gel bands representing break points within the BglII fragment sequence. (B&C) Endo III treated samples (+) and (−) DMSO respectively. Untreated BglII fragments (closed diamonds); endo III treated fragments (open squares). (D&E) Fpg treated samples (+) and (−) DMSO respectively. Untreated BglII fragments (closed diamonds); Fpg treated fragments (open circles). The points on the graphs represent the average percent densitometric band area ± SEM for bands observed in two experiments (panels B & D), or three experiments (panels C & E).

Enzymatic probing of the BglII fragments 5′-end labeled lower strand. (A ) The BglII fragment model for the longest sequence detected. An asterisk indicates the 5′-³²P-end label, and the targeted G is shown in bold. The breakage-maximum is indicated by a lowercase “t”. (B-E) Densitometric quantification of sequencing gel bands representing break points within the BglII fragment sequence. (B&C) Endo III treated samples (+) and (−) DMSO respectively. Untreated BglII fragments (closed diamonds); endo III treated fragments (open squares). (D&E) Fpg treated samples (+) and (−) DMSO respectively. Untreated BglII fragments (closed diamonds); Fpg treated fragments (open circles). The points on the graphs represent the average percent densitometric band area ± SEM for bands observed in two experiments (panels B & D), or three experiments (panels C & E).

Lesion identification. GC/MS analysis of base damage in the same DSB terminated BglII restriction fragment samples used as substrates for repair enzyme probing of base damage. Results are expressed as a ratio of the lesion identified in the damaged DNA sample to the background observed in the undamaged control BglII/MbiI DNA fragment (see Fig. 1). Three types of damage were detected; 8-OH-Ade, 8-OH-Gua and 5-OH-Cyt. 8-OH-Gua was detected in the highest amount in both samples tested. Larger amounts of each of the three lesion types were observed in the (−) DMSO samples compared to the (+) DMSO samples.

Enzyme probing of the BglII fragments 5′-end labeled upper strand. (A) The BglII fragment model depicting the longest sequence detected. An asterisk indicates the 5′-³²P-end label, and the ¹²⁵I-TFO targeted duplex G is bold. The subscripted number permits sequence correlation to those in panels B-F and Fig. 1. The lowercase “a” is the breakage maximum. (B) A typical 20% denaturing PAGE separation of DSB terminated upper strands. Lanes 1 and 2, Maxam & Gilbert “G” and “G+A” sequencing ladders; lanes 3 and 6, ¹²⁵I-DSB terminated BglII fragments produced by irradiation in (+) or (−) DMSO respectively; lanes 4 and 7, endo III treated BglII fragments (+) or (−) DMSO respectively; lanes 5 and 8, Fpg treated BglII fragments (+) or (−) DMSO respectively; lane 9, undamaged duplex 47 bp control fragment; lane 10, endo III treated 47 bp control; lane 11, Fpg treated 47 bp control. Bands representing break points at nucleotides within the target sequence are numbered according to their position with respect to the longest fragment identified, counting up as the breaks progress toward the BglII restriction site. (C-F) Densitometric quantification of sequencing gel bands representing break points within the BglII fragment sequence. (C&D) Endo III treated samples (+) and (−) DMSO respectively. Untreated BglII fragments (closed diamonds); endo III treated fragments (open squares). (E&F) Fpg treated samples (+) and (−) DMSO respectively. Untreated BglII fragments (closed diamonds); Fpg treated fragments (open circles). The points on the graphs represent the average percent densitometric band area ± SEM for bands observed in three experiments (panels C & E), or two experiments (panels D & F).

Enzymatic probing of the BglII fragments 3′-end labeled lower strand. (A) The BglII fragment model for the longest sequence detected. An asterisk indicates the 3′-³²P-end label, and the targeted G is shown in bold. The breakage-maximum is indicated by a lowercase “t”. (B-E) Densitometric quantification of sequencing gel bands representing break points within the BglII fragment sequence. (B&C) Endo III treated samples (+) and (−) DMSO respectively. Untreated BglII fragments (closed diamonds); endo III treated fragments (open squares). (D&E) Fpg treated samples (+) and (−) DMSO respectively. Untreated BglII fragments (closed diamonds); Fpg treated fragments (open circles). The points on the graphs represent the average percent densitometric band area ± SEM for bands observed in three experiments.

Repair enzyme treatment of an internal control fragment. A 49 bp internal control restriction fragment recovered from the ¹²⁵I-DSB linearized pTC27 plasmid (from (+) and (−) DMSO samples) by StuI and SfiI digestion was used to assess non-specific background radiation damage by enzymatic probing with endo III and Fpg. Reactions were run under the same conditions described for repair enzyme probing of the DSB terminated sample fragments (untreated control (C), endo III (E3), formamidopyrimidine-DNA glycosylase (Fpg)).

Identification of increasing base damage frequencies at sites greater than 24 nucleotides upstream from the initial ¹²⁵I decay site. A) Denaturing polyacrylamide gel (20%) showing changes in band densities and formation of short fragments following Fpg treatment of the 5′-³²P-labeled DSB-containing BglII fragment. Lane 1, Maxam & Gilbert sequencing ladder for G; lane 2, untreated DSB-terminated BglII fragments; lane 3, Fpg treated DSB-terminated BglII fragments. The sequencing ladder is labeled at G’s and G₁ represents the longest fragment population observed following DSB induction by ¹²⁵I decay and BglII cleavage. B) Magnified and contrast enhanced lower portion of the gel showing Fpg cleavage-dependent accumulation of small fragments. The estimated size of the fragments and their corresponding position on the sequence is shown in the side bar. Lane notations are the same as in panel A. C) Graphical representation of the loss and gain of the fragments observed in panel A lanes 2 and 3. The G₄ shown in bold represents the base targeted for Hoogsteen base pairing by the ¹²⁵I-dC of the ¹²⁵I-TFO and the lower case ‘a’ represents the DSB breakage frequency maximum.