Rapid DNA-protein cross-linking and strand scission by an abasic site in a nucleosome core particle

Abstract

Apurinic/apyrimidinic (AP) sites are ubiquitous DNA lesions that are highly mutagenic and cytotoxic if not repaired. In addition, clusters of two or more abasic lesions within one to two turns of DNA, a hallmark of ionizing radiation, are repaired much less efficiently and thus present greater mutagenic potential. Abasic sites are chemically labile, but naked DNA containing them undergoes strand scission slowly with a half-life on the order of weeks. We find that independently generated AP sites within nucleosome core particles are highly destabilized, with strand scission occurring ∼60-fold more rapidly than in naked DNA. The majority of core particles containing single AP lesions accumulate DNA-protein cross-links, which persist following strand scission. The N-terminal region of histone protein H4 contributes significantly to DNA-protein cross-links and strand scission when AP sites are produced approximately 1.5 helical turns from the nucleosome dyad, which is a known hot spot for nucleosomal DNA damage. Reaction rates for AP sites at two positions within this region differ by ∼4-fold. However, the strand scission of the slowest reacting AP site is accelerated when it is part of a repair resistant bistranded lesion composed of two AP sites, resulting in rapid formation of double strand breaks in high yields. Multiple lysine residues within a single H4 protein catalyze double strand cleavage through a mechanism believed to involve a templating effect. These results show that AP sites within the nucleosome produce significant amounts of DNA-protein cross-links and generate double strand breaks, the most deleterious form of DNA damage.

Fig. 1.

Independent generation of AP sites within nucleosome core particles. (A) X-ray crystal structure of the α-satellite DNA nucleosome core particle (PDB ID code 1aoi) highlighting positions of AP incorporation. (B) A portion of the α-satellite DNA sequence showing where the AP sites are incorporated (see SI Text for complete DNA sequences). (C) Methods used for independently generating AP sites.

Fig. 2.

Strategy for using gel electrophoresis to analyze the reactivity of AP in nucleosome core particles. (Top) Expected cross-linking and cleavage reactions between an AP site and a lysine residue. (Bottom) Predicted detection of different products using two electrophoresis methods. Denaturing PAGE following histone digestion by proteinase K separates intact nucleosomal DNA (black) from cleaved DNA (red) whether or not the DNA was cross-linked to protein. SDS PAGE separates intact nucleosomal DNA (black), DNA–protein cross-links (DPC, blue), and cleaved DNA that is not cross-linked to protein (SSB, green). Please note that SDS PAGE does not distinguish between DPCs containing cleaved and intact DNA (blue).

Fig. 3.

Reaction of AP₈₉ in a nucleosome core particle (1a) and naked DNA of the same sequence. (A) SDS PAGE analysis describing disappearance of intact DNA containing AP₈₉ in nucleosome core particle 1a and growth of DPC and SSB as a function of time. (B) Comparison of the total amount of strand scission using denaturing PAGE analysis in naked, and nucleosomal DNA from 1a, following protease digestion.

Fig. 4.

Reaction of a bistranded lesion (AP₈₉, AP₂₀₇) in a nucleosome core particle (3a). (A) Disappearance of intact core particle 3a and growth of DPC, SSB, and DSB as a function of time using SDS PAGE (without proteinase K treatment of aliquots). (B) Time dependence of the disappearance of intact nucleosome core particle 3a, and the growth of products, using SDS PAGE following proteinase K digestion of aliquots.

Fig. 5.

Identification of the histone proteins involved in DPC formation with AP sites in nucleosome core particles. (A) DPC formation with AP₈₉ in the presence of wild-type (nucleosome core particle 1a) and tailless H4 protein (nucleosome core particle 1b). (B) DPC formation with AP₂₀₇ in the presence of wild-type (nucleosome core particle 2a) and tailless H4 protein (nucleosome core particle 2b). See also Fig. S4.

Scheme 1.

Method for determining protein(s) cross-linked with AP sites.

Fig. 6.

Identification of the histone proteins involved in DPC formation with AP sites in the bistranded lesion. (A) DPC formation with AP₈₉ (nucleosome core particle 3a, 3b) and AP₂₀₇ (nucleosome core particle 3c, 3d) in the presence of wild-type (3a, 3c) and tailless H4 protein (3b, 3d). (B) Overall DNA–protein cross-link yield, and contribution by AP₈₉ (3a) and AP₂₀₇ (3c) to DPCs in the bistranded substrate when incubated in the presence of NaBH₃CN. Individual contributions by AP₈₉ and AP₂₀₇ were determined by denaturing samples prior to gel electrophoresis.

Scheme 2.

Reaction mechanism for the formation of a DPC and strand break from AP in a nucleosome core particle.

Scheme 3.

Postulated templating effect for the reaction of a bistranded AP lesion with a single histone protein.