Correlation of Thermal Stability and Structural Distortion of DNA Interstrand Cross-Links Produced from Oxidized Abasic Sites with Their Selective Formation and Repair

Abstract

C4'-oxidized (C4-AP) and C5'-oxidized abasic sites (DOB) that are produced following abstraction of a hydrogen atom from the DNA backbone reversibly form cross-links selectively with dA opposite a 3'-adjacent nucleotide, despite the comparable proximity of an opposing dA. A previous report on UvrABC incision of DNA substrates containing stabilized analogues of the ICLs derived from C4-AP and DOB also indicated that the latter is repaired more readily by nucleotide excision repair [Ghosh, S., and Greenberg, M. M. (2014) Biochemistry 53, 5958-5965]. The source for selective cross-link formation was probed by comparing the reactivity of ICL analogues of C4-AP and DOB that mimic the preferred and disfavored cross-links with that of reagents that indirectly detect distortion by reacting with the nucleobases. The disfavored C4-AP and DOB analogues were each more reactive than the corresponding preferred cross-link substrates, suggesting that the latter are more stable, which is consistent with selective ICL formation. In addition, the preferred DOB analogue is more reactive than the respective C4-AP ICL, which is consistent with its more efficient incision by UvrABC. The conclusions drawn from the chemical probing experiments are corroborated by UV melting studies. The preferred ICLs exhibit melting temperatures higher than those of the corresponding disfavored isomers. These studies suggest that oxidized abasic sites form reversible interstrand cross-links with dA opposite the 3'-adjacent thymidine because these products are more stable and the thermodynamic preference is reflected in the transition states for their formation.

Figure 1

Abasic site structures.

Figure 2

Molecular model of C4-AP in a 5′-d(TXT)/(AAA) sequence (X = C4-AP).

Figure 3

Cross-linked DNA substrates used in this study.

Figure 4

Chemical reactivity of DOB ICL analogues in 5′-³²P-b-1–3 with (A) DEPC at A₁₁ relative to A₈, (B) DMS at G₁₃ relative to G₆, and (C) DEPC at X₁₄ relative to A₈ (X = A in 2 and 3, and X = cross-linked A in 1). P values were calculated using the Student’s t test (four replicates).

Figure 5

Chemical reactivity of DOB ICL analogues in 3′-³²P-b-1–3 with DEPC at (A) X₁₅ relative to A₂₃ (X = A in 1 and 3, and X = cross-linked A in 2), (B) A₁₆ relative to A₂₃, and (C) A₁₇ relative to A₂₃. P values were calculated using the Student’s t test (four replicates).

Figure 6

Chemical reactivity of DOB ICL analogues in 5′-³²P-t-1–3 with KMnO₄ at (A) T₄₄ relative to T₃₈ and (B) T₄₅ relative to T₃₈. P values were calculated using the Student’s t test (four replicates).

Figure 7

Chemical reactivity of DOB ICL analogues in 3′-³²P-t-1–3 at (A) X₄₆ relative to G₅₄ (X = T in 3, and X = cross-linked abasic site in 1 and 2) with hydroxyl radical, (B) T₄₇ relative to G₅₄ with KMnO₄, (C) C₄₈ relative to C₅₅ with hydroxylamine, and (D) G₄₉ relative to G₅₄ with DMS. P values were calculated using the Student’s t test (four replicates).

Figure 8

Chemical reactivity of C4-AP ICL analogues in 5′-³²P-b-10–12 upon (A) reaction with DEPC, (B) reaction with KMnO₄, and (C) reaction with DEPC. P values were calculated using the Student’s t test (four replicates).

Figure 9

Chemical reactivity of C4-AP ICL analogues in 3′-³²P-b-8–10 at (A) X₁₈ relative to A₂₈ (X = A in 8 and 10, and X = cross-linked A in 9) with DEPC, (B) T₁₉ relative to T₂₉ with KMnO₄, and (C) A₂₀ relative to A₂₈ with DEPC. P values were calculated using the Student’s t test (four replicates).

Figure 10

Chemical reactivity of C4-AP ICL analogues in 5′-³²P-t-8–10 at (A) A₅₂ relative to G₄₈ with DEPC, (B) T₅₃ relative to T₄₅ with KMnO₄, and (C) A₅₄ relative to G₄₈ with DEPC. P values were calculated using the Student’s t test (four replicates).

Figure 11

Chemical reactivity of C4-AP ICL analogues in 3′-³²P-t-8–10 at (A) V₅₅ relative to A₆₇ (X = T in 10, and X = cross-linked abasic site in 8 and 9) with hydroxyl radical, (B) T₅₆ relative to T₆₈ with KMnO₄, (C) A₅₇ relative to A₆₇ with DEPC, and (D) T₅₈ relative to T₆₈ with KMnO₄. P values were calculated using the Student’s t test (four replicates).

Figure 12

UV melt of (A) DOB ICL analogues (4 and 5) and (B) C4-AP ICL analogues (13 and 14).

Figure 13

Nucleotide by nucleotide comparison of ICL reactivity relative to analogous duplex: (A) DOB-containing ICL and (B) C4-AP-containing ICL.

Scheme 1

ICLs and Stabilized ICLs from C4-AP and DOB

Scheme 2

ICL Positioning from C4-AP and DOB