Probing Enhanced Double-Strand Break Formation at Abasic Sites within Clustered Lesions in Nucleosome Core Particles

Abstract

DNA is rapidly cleaved under mild alkaline conditions at apyrimidinic/apurinic sites, but the half-life is several weeks in phosphate buffer (pH 7.5). However, abasic sites are ∼100-fold more reactive within nucleosome core particles (NCPs). Histone proteins catalyze the strand scission, and at superhelical location 1.5, the histone H4 tail is largely responsible for the accelerated cleavage. The rate constant for strand scission at an abasic site is enhanced further in a nucleosome core particle when it is part of a bistranded lesion containing a proximal strand break. Cleavage of this form results in a highly deleterious double-strand break. This acceleration is dependent upon the position of the abasic lesion in the NCP and its structure. The enhancement in cleavage rate at an apurinic/apyrimidinic site rapidly drops off as the distance between the strand break and abasic site increases and is negligible once the two forms of damage are separated by 7 bp. However, the enhancement of the rate of double-strand break formation increases when the size of the gap is increased from one to two nucleotides. In contrast, the cleavage rate enhancement at 2-deoxyribonolactone within bistranded lesions is more modest, and it is similar in free DNA and nucleosome core particles. We postulate that the enhanced rate of double-strand break formation at bistranded lesions containing apurinic/apyrimidinic sites within nucleosome core particles is a general phenomenon and is due to increased DNA flexibility.

Figure 1

General structural features of a nucleosome core particle showing one gyre of the DNA (Protein Data Bank entry 1kx5). Nucleotide positions at which abasic sites are incorporated are colored red; clockwise from the top are positions 73, 205, and 89, respectively.

Figure 2

Representative plots of the effect of a nick or one-nucleotide gap (containing various termini) in the opposing strand on the rate of disappearance of AP₂₀₅ in a NCP. The numbers 1–5 correspond to substrate numbers in Table 1.

Figure 3

Representative plots of the effect of distance from a one-nucleotide or the presence of a two-nucleotide gap in the opposing strand on the rate of disappearance of AP₈₉ in a NCP. The numbers 6–10 correspond to substrate numbers in Table 2.

Figure 4

Representative plots of the effect of a nick or one-nucleotide gap in the opposing strand on the rate of disappearance of an abasic site at the dyad axis (AP₇₃) in a NCP. The numbers 1–5 correspond to substrate numbers in Table 1. The numbers 11–13 correspond to substrate numbers in Table 3.

Figure 5

Representative plots of the effect of a nick or one-nucleotide gap in the opposing strand on the rate of disappearance of 2-deoxyribonolactone at SHL 1.5 (L₈₉) in a NCP. The numbers 14–16 correspond to substrate numbers in Table 4.

Figure 6

Representative plots of the effect of a nick or one-nucleotide gap in the opposing strand on the rate of disappearance of 2-deoxyribonolactone at SHL 1.5 (L₇₃) in a NCP. The numbers 17–19 correspond to substrate numbers in Table 5.

Scheme 1