Roles of homologous recombination in response to ionizing radiation-induced DNA damage

Abstract

Purpose: Ionizing radiation induces a vast array of DNA lesions including base damage, and single- and double-strand breaks (SSB, DSB). DSBs are among the most cytotoxic lesions, and mis-repair causes small- and large-scale genome alterations that can contribute to carcinogenesis. Indeed, ionizing radiation is a 'complete' carcinogen. DSBs arise immediately after irradiation, termed 'frank DSBs,' as well as several hours later in a replication-dependent manner, termed 'secondary' or 'replication-dependent DSBs. DSBs resulting from replication fork collapse are single-ended and thus pose a distinct problem from two-ended, frank DSBs. DSBs are repaired by error-prone nonhomologous end-joining (NHEJ), or generally error-free homologous recombination (HR), each with sub-pathways. Clarifying how these pathways operate in normal and tumor cells is critical to increasing tumor control and minimizing side effects during radiotherapy.

Conclusions: The choice between NHEJ and HR is regulated during the cell cycle and by other factors. DSB repair pathways are major contributors to cell survival after ionizing radiation, including tumor-resistance to radiotherapy. Several nucleases are important for HR-mediated repair of replication-dependent DSBs and thus replication fork restart. These include three structure-specific nucleases, the 3' MUS81 nuclease, and two 5' nucleases, EEPD1 and Metnase, as well as three end-resection nucleases, MRE11, EXO1, and DNA2. The three structure-specific nucleases evolved at very different times, suggesting incremental acceleration of replication fork restart to limit toxic HR intermediates and genome instability as genomes increased in size during evolution, including the gain of large numbers of HR-prone repetitive elements. Ionizing radiation also induces delayed effects, observed days to weeks after exposure, including delayed cell death and delayed HR. In this review we highlight the roles of HR in cellular responses to ionizing radiation, and discuss the importance of HR as an exploitable target for cancer radiotherapy.

Keywords: DNA double-strand breaks; DNA repair; cancer radiotherapy; homologous recombination; ionizing radiation; replication stress.

Figure 1.

Overview of ionizing radiation effects on DNA and DSB repair pathways. Three types of ionizing radiation are currently used therapeutically. Photons are massless energy, protons are low mass particles with a single positive charge, and carbon ions have six positively charged protons and six neutrons. Protons and neutrons are shown by red and grey circles, respectively. Radiation can ionize DNA directly or indirectly via production of ROS (principally H₂O) that attacks DNA. Five major classes of DNA damage that result are diagrammed, including DSBs, the principal cytotoxic lesions that are repaired by NHEJ and HR. Dominant ionization mechanisms and DSB repair pathways are indicated by thick arrows.

Figure 2.

HR-mediated DSB repair mechanisms. (A) DSB repair by conservative HR can proceed by two-strand invasion (left) of a homologous donor duplex, producing a double HJ intermediate that can be resolved into crossover or non-crossover gene conversion products. Dissolution of the double HJ intermediate can occur by convergence of HJs by branch migration (open arrows) to yield a non-crossover product. HJ resolution by cleavage/rejoining (filled arrows), leads to non-crossover products if cleaved in the same ‘horizontal’ or ‘vertical’ sense (bottom left), or to crossover products if cleaved in opposite senses (bottom right). SDSA (right) involves a single end invasion, repair synthesis across the DSB, release from the donor, and reannealing to the non-invading strand, followed by gap filling and DNA ligation. (B) SSA shown between linked, direct repeats. Resection from broken ends exposes ssDNA in flanking homologous sequences (shaded arrows) that anneal. Flap trimming and gap filling completes repair, with one repeat and intervening sequence deleted, yielding non-conservative repair products.

Figure 3.

HR-mediated DSB repair enzymology. Resection by indicated nucleases prevents DSB repair by classical or alternative NHEJ, exposing 3’ ssDNA tails that are first coated by RPA and then RAD51, an exchange facilitated by RAD51 paralogs, BRCA2, RAD52, RAD54 and other factors. RAD51-ssDNA filaments invade homologous duplex DNA, and RAD51 dissociates to allow repair synthesis. In this SDSA reaction, the invading strand is released to reanneal with the second broken (resected) end. Further repair synthesis fills remaining gaps and ligation completes repair.

Figure 4.

HR-mediated repair and restart of stressed replication forks. Ionizing radiation induces single-strand lesion (shown here as ring-opened base, red) that blocks a replication fork. MUS81 cleavage yields a one-ended DSB (EEPD1 cleaves in the opposite polarity; see Figure 5). Resection of the broken end initiates RAD51-mediated strand invasion of the sister chromatid, followed by repair synthesis, and release/reannealing of the invading strand, as shown in Figure 3. Failure to resect the one-ended DSB may lead to genome rearrangements via NHEJ-mediated joining of one-ended DSBs from different collapsed forks.

Figure 5.

Polarity of fork cleavage may influence timing of fork restart. (A) 5’ or 3’ fork cleavage by EEPD1 or MUS81/EME2, respectively, can initiate HR-mediated fork repair and restart via steps as shown in Figure 3. (B) 3’ fork cleavage by MUS81/EME2 forces strand invasion into sister chromatid produced by lagging strand DNA synthesis, which has discontinuous Okazaki fragments adjacent to the fork and mature, continuous duplex DNA further from the fork. Limited resection may result in failed invasion into discontinuous region (red X), but more extensive resection, requiring more time, allows successful invasion into mature duplex DNA. 5’ cleavage by EEPD1 may speed fork restart as less resection is needed because strand invasion can occur anywhere along the continuous, leading strand sister chromatid (see panel A).