Abasic and oxidized abasic site reactivity in DNA: enzyme inhibition, cross-linking, and nucleosome catalyzed reactions

Abstract

Abasic lesions are a family of DNA modifications that lack Watson-Crick bases. The parent member of this family, the apurinic/apyrimidinic lesion (AP), occurs as an intermediate during DNA repair, following nucleobase alkylation, and from random hydrolysis of native nucleotides. In a given day, each cell produces between 10000 and 50000 AP lesions. A variety of oxidants including γ-radiolysis produce oxidized abasic sites, such as C4-AP, from the deoxyribose backbone. A number of potent, cytotoxic antitumor agents, such as bleomycin and the enediynes (e.g., calicheamicin, esperamicin, and neocarzinostatin) also lead to oxidized abasic sites in DNA. The absence of Watson-Crick bases prevents DNA polymerases from properly determining which nucleotide to incorporate opposite abasic lesions. Consequently, several studies have revealed that (oxidized) abasic sites are highly mutagenic. Abasic lesions are also chemically unstable, are prone to strand scission, and possess electrophilic carbonyl groups. However, researchers have only uncovered the consequences of the inherent reactivity of these electrophiles within the past decade. The development of solid phase synthesis methods for oligonucleotides that both place abasic sites in defined positions and circumvent their inherent alkaline lability has facilitated this research. Chemically synthesized oligonucleotides containing abasic lesions provide substrates that have allowed researchers to discover a range of interesting chemical properties of potential biological importance. For instance, abasic lesions form DNA-DNA interstrand cross-links, a particularly important family of DNA damage because they block replication and transcription absolutely. In addition, bacterial repair enzymes can convert an interstrand cross-link derived from C4-AP into a double-strand break, the most deleterious form of DNA damage. Oxidized abasic lesions can also inhibit DNA repair enzymes that remove damaged nucleotides. DNA polymerase β, an enzyme that is irreversibly inactivated, is vitally important in base excision repair and is overproduced in some tumor cells. Nucleosome core particles, the monomeric components that make up chromatin, accentuate the chemical instability of abasic lesions. In experiments using synthetic nucleosome core particles containing abasic sites, the histone proteins catalyze strand cleavage at the sites that incorporate these lesions. Furthermore, in the presence of the C4-AP lesion, strand scission is accompanied by modification of the histone protein. The reactivity of (oxidized) abasic lesions illustrates how seemingly simple nucleic acid modifications can have significant biochemical effects and may provide a chemical basis for the cytotoxicity of the chemotherapeutic agents that produce them.

Figure 1

Effects on ICL formation when C4-AP is opposite thymidine (16). A. ICL rescue as a function of [adenine]. B. Effects of various purines on ICL yield.

Scheme 1

Examples of DNA abasic sites.

Scheme 2

Synthetic methods for preparing oligonucleotides containing AP sites.

Scheme 3

Synthetic methods for preparing oligonucleotides containing C4-AP and DOB lesions.

Scheme 4

Synthetic methods for preparing oligonucleotides containing L lesions.

Scheme 5

Overview of base excision repair (BER) of AP sites.

Scheme 6

Removal of AP lesion by BER.

Scheme 7

Reaction of bifunctional BER glycosylases with AP.

Scheme 8

Long patch base excision repair.

Scheme 9

Inactivation of Pol β by DOB.

Scheme 10

DNA interstrand cross-link formation by AP.

Scheme 11

DNA interstrand cross-link formation by C4-AP.

Scheme 12

NER of an ICL.

Scheme 13

DSB formation of NER misrepair.

Scheme 14

AP cleavage in a nucleosome core particle.

Scheme 15

C4-AP cleavage in a nucleosome core particle.