Investigating the reactivity of an abasic lesion within nucleosomes and probing the interactions between histone tails and nucleosomal DNA in nucleosome core particles

DNA abasic lesion;DOB;nucleosome;DNA-protein cross-links;nucleosome core particle;histone tails;histone tail-DNA interactions;Chemistry

Weng, LiweiGreenberg, Marc M. ;

Johns Hopkins University

关键词: DNA abasic lesion;    DOB;    nucleosome;    DNA-protein cross-links;    nucleosome core particle;    histone tails;    histone tail-DNA interactions;    Chemistry;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/37902/WENG-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

DNA lesions are the source of the cytotoxicity of various antitumor reagents and γ-radiolysis. Studying the reactivity of these lesions and their effects in biological processes may provide the chemical basis for anticancer treatment and shed light on the rational design of novel drugs. The oxidized abasic lesion 5’-(2-phosphoryl-1,4-dioxobutane) (DOB) is produced concomitantly with a strand break via C5;;-hydrogen atom abstraction by a variety of DNA-damaging agents. Herein, we report that the reactivity of DOB is distinct in nucleosomes from that in free DNA. DOB undergoes cleavage at a significantly higher rate in NCPs compared to free DNA, exhibiting a half-life of 8.5-16.8 min depending upon the sites in which DOB is generated. The rate constant of DOB decomposition is faster than its expected rate of repair in cells. DOB decomposition results in a pyrrolone modification of the histone tail. Furthermore, DOB decomposition within the linker DNA that connects two adjacent nucleosomes is also accelerated, albeit to a lesser extent than that within NCPs. The pyrrolone modification was observed in the histone H3 as a result of the interactions of the H3 tail interacting with the lesion in the linker region. The unnatural modification resulting from reaction with the DNA lesion may have profound biological consequences. The interactions between histone tails and nucleosomal DNA play significant roles in chromatin assembly and the regulation of gene expression. Recent studies including the one presented above revealed that residues on the N-terminal tails of histone proteins are intensively involved in catalyzing the decomposition of DNA lesions at specific sites within NCPs. Despite their importance, such interactions are not well defined due to the mobility of histone tails and the transient nature of the interactions. To gain insight into such interactions, we developed a new DNA-protein cross-linking (DPC) method by taking advantage of the reaction between a modified electrophilic nucleotide (2) and the nucleophilic residues in histone tails. Phenyl selenide 1 rapidly produces 2 upon mild oxidation, which reacts with nucleophilic amino acid side chains. The facile reaction and high DPC yields produced from 2 enabled both product and kinetic analysis, which ranked the contributions by individual and/or groups of amino acids (His18 > Lys16 > Lys20 ~ Lys8,12 > Lys5) that react with 2 at position 89 within the NCP, a hot spot for DNA damage. In addition, photolysis of 1 produced ICLs in free DNA and NCPs. The reexamination of photochemistry of 1 revealed that a carbocation intermediate (19) is responsible for the DNA ICL formation.