The mechanism of DNA double-strand break (DSB) resection in human cells (original) (raw)

DNA Double-Strand Break Rejoining in Complex Normal Tissues

International Journal of Radiation Oncology*Biology*Physics, 2008

Purpose: The clinical radiation responses of different organs vary widely and likely depend on the intrinsic radiosensitivities of their different cell populations. Double-strand breaks (DSBs) are the most deleterious form of DNA damage induced by ionizing radiation, and the cells' capacity to rejoin radiation-induced DSBs is known to affect their intrinsic radiosensitivity. To date, only little is known about the induction and processing of radiation-induced DSBs in complex normal tissues. Using an in vivo model with repair-proficient mice, the highly sensitive gH2AX immunofluorescence was established to investigate whether differences in DSB rejoining could account for the substantial differences in clinical radiosensitivity observed among normal tissues. Methods and Materials: After whole body irradiation of C57BL/6 mice (0.1, 0.5, 1.0, and 2.0 Gy), the formation and rejoining of DSBs was analyzed by enumerating gH2AX foci in various organs representative of both early-responding (small intestine) and late-responding (lung, brain, heart, kidney) tissues. Results: The linear dose correlation observed in all analyzed tissues indicated that gH2AX immunofluorescence allows for the accurate quantification of DSBs in complex organs. Strikingly, the various normal tissues exhibited identical kinetics for gH2AX foci loss, despite their clearly different clinical radiation responses. Conclusion: The identical kinetics of DSB rejoining measured in different organs suggest that tissue-specific differences in radiation responses are independent of DSB rejoining. This finding emphasizes the fundamental role of DSB repair in maintaining genomic integrity, thereby contributing to cellular viability and functionality and, thus, tissue homeostasis. Ó

The cellular response to general and programmed DNA double strand breaks

DNA Repair, 2004

DNA double strand breaks (DSBs) are among the most dangerous lesions that can occur in the genome of eukaryotic cells. Proper repair of chromosomal DSBs is critical for maintaining cellular viability and genomic integrity and, in multi-cellular organisms, for suppression of tumorigenesis. Thus, eukaryotic cells have evolved specialized and redundant molecular mechanisms to sense, respond to, and repair DSBs. In this chapter, we provide an overview of the progress that has been made over the last decade in elucidating the identity and function of components that participate in the cellular response to chromosomal DSBs. Then, we discuss, in more depth, the response to DSBs that occur in the context of the V(D)J recombination and IgH class switch recombination reactions that occur in cells of the lymphocyte lineage.

Generation of DNA Double-strand Breaks by Two Independent Enzymatic Activities in Nuclear Extracts

Journal of Molecular Biology, 2005

We have reported the existence in rat nuclear extracts of a specific cleavage activity on a DNA fragment containing the human minisatellite MsH42 region (minisatellite plus its flanking sequences). Here, we have developed a system to analyse the nature of the cleavage products from the MsH42 region generated by the nuclear extracts. Our results demonstrated the formation of DNA double-strand breaks (DSB) in the MsH42 region by two different enzymatic activities, and that their distribution along this fragment changes depending on the presence of Mg 2C . In the assays with Mg 2C , the DSB were located in the minisatellite and its 3 0 -flanking region, showing preference for G-rich stretches. Oligonucleotide mutagenesis analysis confirmed that this enzymatic activity has a strong preference for G-tracts and that the recognition site is polarized towards the 3 0 end. Moreover, this activity cuts GC palindromes efficiently. In contrast, in the experiments without Mg 2C , most DSB were mapped within the minisatellite flanking sequences. The analysis with oligonucleotides showed that G-tracts are recognized by this endonuclease activity, but with differences in the cleavage behaviour with respect to the reactions observed with Mg 2C . The existence of two separate activities (Mg 2Cdependent and Mg 2C -independent) for the production of DSB was confirmed by analysing the effect of EGTA, N-ethyl maleimide, ionic strength, and by preincubations of the nuclear extracts at different temperatures. The tissue distribution of both DSB-producing activities was also different. The in vitro system used in the present work may be a useful tool for studying the formation of DSB and for investigation of the mechanisms of DNA repair.

Double-Strand Break Formation during Nucleotide Excision Repair of a DNA Interstrand Cross-Link

Biochemistry, 2009

The DNA interstrand cross-link (ICL) resulting from the C4′-oxidized abasic site (C4-AP) is a unique clustered lesion comprised of a cross-link adjacent to a nick. The ICL is a substrate for the UvrABC nucleotide excision repair system. The strand containing the nick is preferentially incised, but the nick influences the cleavage sites. Moreover, in approximately 15% of the molecules the strand opposite the nick is incised, resulting in a more toxic double strand break. This is the first example in which an interstrand cross-link is converted by nucleotide excision misrepair into a more deleterious double strand break. DNA double-strand breaks (DSBs) are considered to be the most dangerous form of DNA damage. Even one DSB is sufficient to kill a cell (1). Much of this danger arises from the intrinsic difficulty in repairing a severed DNA molecule. Erroneous rejoining of DNA DSBs leads to genomic instability, which can lead to tumorgenesis (2). DNA DSBs are formed by endogenous reactive oxygen species and mechanical chromosomal stress. DSBs also result from exogenous sources such as ionizing radiation and the chemotherapeutic agent bleomycin due to the accumulation of single-strand breaks (SSBs) in close proximity to one another (4, 5). DNA interstrand crosslinks (ICLs) are also highly deleterious DNA lesions. ICLs are absolute blocks to replication and transcription and may also give rise to DSBs during repair. Studies in cells and lysates indicate that DSBs are produced from psoralen cross-links during repair (6,7). However, the protein(s) responsible have not been identified. The C4′-oxidized abase site (C4-AP) is produced by a variety of DNA damaging agents (8). It is most often associated with bleomycin reactions where it accounts for as much as 40% of the products (9). We recently reported that C4-AP forms stable ICLs in DNA following its elimination to 1 (Scheme 1) (10). Elimination is catalyzed by an opposing 2′-deoxyadenosine and by DNA lyases such as endonuclease III (Nth). The unsaturated aldehyde (1) reacts with dA or dC in the opposite strand to form ICLs (e.g. 2) (10,11). DNA damage of this type is referred to as a complex or clustered lesion. Clustered lesions are defined by the presence of two or more damage sites within ~1.5 helical turns (12). Although it has been suggested that clustered lesions can be converted to DSBs during repair (13,14), we provide the first direct evidence that an ICL (2) is converted to a DSB during nucleotide excision repair (NER). In bacteria, the three-component endonuclease UvrABC recognizes and incises damage including cross-links in distorted DNA (15). The UvrA 2 B complex is responsible for locating