Abstract
Clusters of DNA damage, also called multiply damaged sites (MDS), are a signature of ionizing radiation exposure. They are defined as two or more lesions within one or two helix turns, which are created by the passage of a single radiation track. It has been shown that the clustering of DNA damage compromises their repair. Unresolved repair may lead to the formation of double-strand breaks (DSB) or the induction of mutation. We engineered three complex MDS, comprised of oxidatively damaged bases and a one-nucleotide (1 nt) gap (or not), in order to investigate the processing and the outcome of these MDS in yeast Saccharomyces cerevisiae. Such MDS could be caused by high linear energy transfer (LET) radiation. Using a whole-cell extract, deficient (or not) in base excision repair (BER), and a plasmid-based assay, we investigated in vitro excision/incision at the damaged bases and the mutations generated at MDS in wild-type, BER, and translesion synthesis-deficient cells. The processing of the studied MDS did not give rise to DSB (previously published). Our major finding is the extremely high mutation frequency that occurs at the MDS. The proposed processing of MDS is rather complex, and it largely depends on the nature and the distribution of the damaged bases relative to the 1 nt gap. Our results emphasize the deleterious consequences of MDS in eukaryotic cells.
Highlights
The spatial distribution and reparability of DNA damage are key parameters in the triggering of lethal or mutagenic events in cells
Clustered DNA damage, so-called Multiply Damaged Sites (MDS), are essentially induced by ionizing radiation; that is, when a single radiation track hits the DNA double helix associated with its water molecules, causing clusters of ionizations that lead to clusters of lesions, at the nanometer scale [1,2,3]
Using a biochemical approach with human and rodent cell extracts on the same constructs, we previously showed that the base excision repair (BER) of an individual lesion within these complex MDS was impaired, largely depending on the nature and distribution of base damage and gap, and on the availability of repair proteins, limiting doublestrand breaks (DSB) formation [42,43]
Summary
The spatial distribution and reparability of DNA damage are key parameters in the triggering of lethal or mutagenic events in cells. Clustered DNA damage is defined as two or more DNA lesions distributed on both strands within one or two helix turns, comprising a combination of oxidized bases, apurinic/apyrimidinic sites (AP sites), and single- or double-strand breaks (SSB and DSB, respectively). The detection of clustered DNA damage has been reported in isolated DNA and in cells after exposure to ionizing radiation, including low-energy electrons, or even by H2O2-induced oxidative stress ([4,5,6,10,11,12,13,14], reviewed in [15]). High LET radiation, which induces clustered DNA damage at a much higher frequency than γ-rays, produces the exact same oxidatively damaged bases as γ-rays and with the same occurrence, only with a different distribution. Clustered DNA damage is considered as the most deleterious lesion induced by ionizing radiation
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