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 crosslinks 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.chromatin | oxidative damage | histone modification E xogenously and endogenously produced agents continuously damage DNA. More than 70 types of damage have been identified, and significant effort is expended to determine which of these lesions are biologically important (1, 2). The apurinic/apyrimidinic (AP) site is a ubiquitous form of DNA damage that is produced in excess of 10,000 lesions per cell each day (3). This highly mutagenic lesion results from spontaneous hydrolysis of native and damaged nucleotides. AP sites are also formed as intermediates during base excision repair of alkylated and oxidized nucleotides and are themselves removed by multiple enzymatic pathways (4, 5). In mammalian cells incision of the AP's 5′-phosphate by Ape1, followed by excision of the resulting 5′-terminal 2′-deoxyribose phosphate by DNA polymerase β is the major pathway for removing the lesion. Redundant repair pathways are reflective of the high mutagenic potential and large quantities of AP produced and are indicative of their physiological significance. Mammalian cells lacking Ape1, the major base excision repair protein responsible for incising AP sites, are embryonic lethal (6). Recent observations reinforce the perception that AP sites are cytotoxic. For instance, formation of bursts of AP sites by the natural product leinamycin is postulated to be the source of this antitumor agent's cy...

Functional relevance of the histone γH2Ax in the response to DNA damaging agents

“…3A). A PARP-1-dependent BER is therefore a major pathway in cells damaged by temozolamide, and γH2Ax is required for repair of a smaller fraction of the total damage (25). Inhibiting PARP-1 slightly sensitized knockout but not mutant cells to low doses of rotenone ( Fig.…”

“…BER transiently generates abasic sites that can be converted to DSBs in chromatin (25). The reduced survival of H2Ax knockout and mutant cells exposed to rotenone indicates that poisoning of mitochondrial complex I (23) generates sufficient ROS to reach the nucleus, activate ATM kinase, and cause DNA damage that requires H2Ax for survival.…”

“…These results are consistent with our previous dem- radiation or endogenously generated ROS from mitochondrial poisoning. ‡ Recent work has shown that a fraction of the AP sites generated during BER can react in nucleosomes to produce DSBs (25). Rot Temoz UV UVcaf Cispl MitC C Fig.…”

Ku Antigen Interaction with Apurinic/Apyrimidinic Sites: Nonhomologous End Joining Vs Base Excision Repair

“…Interaction of proteins with AP sites via a Schiff base intermediate is characteristic of AP lyase activity, intrinsic for the BER proteins -bifunctional DNA glycosylases, which incise DNA backbone via β-or β, δ-elimination [17]. On the other hand, interaction of AP DNA with mammalian proteins not formally involved in BER, but capable of AP site cleavage, is well documented [18][19][20][21]. Interestingly, the interactions of proteins with AP sites via a Schiff-base intermediate without the concomitant DNA incision are also reported [22,23].…”

DNA damage and base excision repair : an important role during zebrafish embryogenesis

“…Sources of DNA damage agents can be exogenous, such as ultraviolet light generating bulky cyclobutane dimers from adjacent pyrimidines and ionizing radiation that damages bases and breaks phosphodiester bonds (Friedberg et al 2006). Alternatively, damage agents can be endogenous, for example, uracil as dUTP, an analogue of thymine, is routinely incorporated into DNA during DNA synthesis or arises through spontaneous deamination of cytosine (Zharkov, Mechetin, and Nevinsky 2010), while spontaneous hydrolysis of nucleotides generates highly deleterious apurinic/apyrimidinic or abasic (AP) sites (Sczepanski et al 2010). However, arguably the most important endogenous source of damage, both quantitatively and qualitatively, is the ongoing generation of reactive oxygen species (ROS).…”