The modulation of topoisomerase I-mediated DNA cleavage and the induction of DNA-topoisomerase I crosslinks by crotonaldehyde-derived DNA adducts

Abstract

Crotonaldehyde is a representative alpha,beta-unsaturated aldehyde endowed of mutagenic and carcinogenic properties related to its propensity to react with DNA. Cyclic crotonaldehyde-derived deoxyguanosine (CrA-PdG) adducts can undergo ring opening in duplex DNA to yield a highly reactive aldehydic moiety. Here, we demonstrate that site-specifically modified DNA oligonucleotides containing a single CrA-PdG adduct can form crosslinks with topoisomerase I (Top1), both directly and indirectly. Direct covalent complex formation between the CrA-PdG adduct and Top1 is detectable after reduction with sodium cyanoborohydride, which is consistent with the formation of a Schiff base between Top1 and the ring open aldehyde form of the adduct. In addition, we show that the CrA-PdG adduct alters the cleavage and religation activities of Top1. It suppresses Top1 cleavage complexes at the adduct site and induces both reversible and irreversible cleavage complexes adjacent to the CrA-PdG adduct. The formation of stable DNA-Top1 crosslinks and the induction of Top1 cleavage complexes by CrA-PdG are mutually exclusive. Lastly, we found that crotonaldehyde induces the formation of DNA-Top1 complexes in mammalian cells, which suggests a potential relationship between formation of DNA-Top1 crosslinks and the mutagenic and carcinogenic properties of crotonaldehyde.

Figure 1.

Covalent trapping of the DNA–protein crosslink formed between Top1 and a double-stranded DNA oligonucleotide containing a CrA-PdG adduct. (A) Shown is the sequence of the 22-bp oligonucleotide used in the study. The position of the CrA-PdG adduct is indicated by a box and asterisk on the lower strand. The positions of the high affinity Top1 cleavage site and the ³²P-radiolabel are also shown. (B) Both single-stranded (ss) and double-stranded (ds) control (WT) or R/S-CrA-PdG (R/S) adducted oligonucleotides were incubated at 25°C for 15 min in the presence or absence of Top1 (250 nM). NaCNBH₃ (50 mM) was added to reactions as indicated. (C) Reactions were carried out exactly as described in (B) with the double-stranded R/S-CrA-PdG (R/S) adducted oligonucleotide in the presence of increasing amounts of Top1 (25, 50, 100, 250 and 500 nM) and the presence of NaCNBH₃ (50 mM). All reaction products were resolved through SDS–PAGE (4–20%) analysis. Arrows indicate the positions of the DNA–Top1 crosslink and the free DNA substrate.

Figure 2.

DNA–Top1 crosslinking capacity of the isolated diastereomeric R- and S-CrA-PdG adducts. (A) Chemical structures of the crotonaldehyde derived R- and S-CrA-PdG adducts. (B) Molecular model and NMR structrure of the R- and S-CrA-PdG adducts, respectively, showing the orientations of the reactive aldehyde moieties and α-carbon methyl groups within the minor groove of the DNA. DNA shown in blue; CrA portion of adduct shown in green; oxygen from the aldehyde moiety shown in red. (C) Double-stranded (ds) control (WT), R/S-, R-, or S-CrA-PdG adducted oligonucleotides were incubated with increasing amounts of Top1 (50 and 250 nM) at 25°C for 15 min in the presence of NaCNBH₃ (50 mM). (D) Double-stranded R- and S-CrA-PdG adducted oligonucleotides were incubated with Top1 (250 nM) at 25°C for 1, 2, 5, 10 and 15 min in the presence of NaCNBH₃ (50 mM). All reaction products were resolved through SDS–PAGE (4–20%) analysis.

Figure 3.

Induction of new Top1-mediated DNA cleavage sites on the DNA strand opposite to the R/S-CrA-PdG adduct. (A) Double-stranded (ds) control (WT) or R/S-CrA-PdG (R/S) adducted oligonucleotides were 3′-end labeled on the scissile strand (upper) with α-³²P ddATP (shown as ³²P-A in B). DNA was then reacted with Top1 and camptothecin in the presence or absence of NaCNBH₃ (50 mM). The size of the fragments generated by Top1 is indicated to the right. Asterisks indicate the positions of new Top1-mediated DNA cleavage sites induced by the CrA-PdG adduct. (B) Summary of Top1-mediated DNA cleavage sites from (A).

Figure 4.

Reversibility of the CrA-PdG adduct-induced Top1 cleavage complexes. (A) Double-stranded (ds) control (WT) or R/S-CrA-PdG (R/S) adducted oligonucleotides were reacted with Top1 in the presence or absence of 1 µM camtothecin at 25°C for 20 min. DNA cleavage was reversed by adding 0.35 M NaCl and monitored over time. (B) Reactions carried out as in (A) were quantified and the percentages of Top1-mediated cleavage products remaining after salt reversal are represented as semi-log plots.

Figure 5.

Detection of DNA–Top1 covalent complexes by crotonaldehyde in cultured mammalian cells. (A) MCF7 cells were treated with 100 μM of crotonaldehyde (CrA) for 6 h. Equal numbers of cells were lyzed and separated by CsCl gradients centrifugation. Fractions were collected from the bottom of the tubes (numbered 1–15). The presence of Top1 was assayed in each of the gradient fractions by western blotting with a Top1 monoclonal antibody. Cellular DNA was contained in fractions 3–7, which corresponds to the DNA–Top1 complexes. (B) MCF7 cells were treated with 0.1 μM CPT or 10 mM CrA for 1 h. All of the DNA-containing fractions (3–7) were collected from the CsCl gradient, pooled together, serial diluted and then blotted with the Top1 monoclonal antibody.

Figure 6.

Summary of DNA–Top1 complex formation by (A) unmodified DNA substrate and (B–C) CrA-PdG adducted DNA substrate. Boxed nucleotide, site of adduct; open triangles, no cleavage; closed triangles, Top1 cleavage site. (A) In the unmodified substrate, Top1 cleavage complexes are trapped by CPT at site 13. Sites 17 and 18 are undetectable. (B) In the CrA-PdG adducted substrate, site 13 is suppressed and two cleavage sites are induced; site 17 is CPT-independent and site 18 is trapped by CPT. (C) Upon reduction by NaCNBH3, Top1 forms an indirect crosslink with the DNA via the CrA-PdG adduct and the formation of Top1 cleavage complexes is inhibited.