Novel Biological Approaches for Testing the Contributions of Single DSBs and DSB Clusters to the Biological Effects of High LET Radiation

Abstract

The adverse biological effects of ionizing radiation (IR) are commonly attributed to the generation of DNA double-strand breaks (DSBs). IR-induced DSBs are generated by clusters of ionizations, bear damaged terminal nucleotides, and frequently comprise base damages and single-strand breaks in the vicinity generating a unique DNA damage-clustering effect that increases DSB "complexity." The number of ionizations in clusters of different radiation modalities increases with increasing linear energy transfer (LET), and is thought to determine the long-known LET-dependence of the relative biological effectiveness (RBE). Multiple ionizations may also lead to the formation of DSB clusters, comprising two or more DSBs that destabilize chromatin further and compromise overall processing. DSB complexity and DSB-cluster formation are increasingly considered in the development of mathematical models of radiation action, which are then "tested" by fitting available experimental data. Despite a plethora of such mathematical models the ultimate goal, i.e., the "a priori" prediction of the radiation effect, has not yet been achieved. The difficulty partly arises from unsurmountable difficulties in testing the fundamental assumptions of such mathematical models in defined biological model systems capable of providing conclusive answers. Recently, revolutionary advances in methods allowing the generation of enzymatic DSBs at random or in well-defined locations in the genome, generate unique testing opportunities for several key assumptions frequently fed into mathematical modeling - including the role of DSB clusters in the overall effect. Here, we review the problematic of DSB-cluster formation in radiation action and present novel biological technologies that promise to revolutionize the way we address the biological consequences of such lesions. We describe new ways of exploiting the I-SceI endonuclease to generate DSB-clusters at random locations in the genome and describe the possible utility of Zn-finger nucleases and of TALENs in generating DSBs at defined genomic locations. Finally, we describe ways to harness the revolution of CRISPR/Cas9 technology to advance our understanding of the biological effects of DSBs. Collectively, these approaches promise to improve the focus of mathematical modeling of radiation action by providing testing opportunities for key assumptions on the underlying biology. They are also likely to further strengthen interactions between experimental radiation biologists and mathematical modelers.

Keywords: CRISPR/Cas9; DSB clusters; DSB repair; I-SceI; RBE; high-LET; radiation effects.

Figure 1

Background information on I-SceI based constructs. (A) Top: recognition sequence of I-SceI endonuclease. Note the generation of 3′ 4-bp overhangs. Bottom: domain structure of an expression vector, pCMV3xnls-I-SceI, used frequently in transient transfection experiments to express I-SceI (85). Note the three NLS sites that ensure the nuclear localization of the expressed enzyme. (B) Schematic representation of the MSGneo2S12His neomycin reporter vector developed to specifically analyze repair of the I-SceI DSB by HRR (86). (C) Schematic representation of the DR-GFP vector developed to specifically analyze repair of the I-SceI DSB by HRR (87). The GFP signal allows analysis by flow cytometry 1–3 days after transfection. (D) Schematic representation of reporter constructs utilizing the Pem1 intron and the Ad2 exon elements, and specifically developed to analyze repair of the I-SceI-DSB by HRR and c-NHEJ, respectively (88).

Figure 2

Outline of reporter constructs developed in the laboratory of Dr. J. Stark (90) to analyze efficiency of I-SceI-DSB-processing by different DSB repair pathways in human cells. (A) DR-GFP construct for analyzing HRR. (B) EJ5-GFP for analyzing c-NHEJ (C) SA-GFP construct for analyzing single-strand annealing (SSA) and (D) EJ2-GFP construct for analyzing alt-EJ.

Figure 3

(A) Recognition sequences of I-PpoI (top) and AsiSI (bottom) endonucleases (see text for details). Note that both endonucleases generate 3′ overhangs. (B) The inducible system of AsiSI endonuclease developed in the laboratory of Dr. Legube (102). The DIvA cell line expresses a form of the AsiSI endonuclease fused to estrogen receptor (ER) and the auxin-inducible degron (AID). The enzyme sequesters under normal conditions in the cytoplasm unable to reach the nucleus and thus to induce DSBs. Administration of tamoxifen (4-OHT) causes efficient translocation of the enzyme to the cell nucleus and the induction of DSBs (top part of the schematic). In this system, the endonuclease activity of AsiSI can be rapidly turned off by removing 4-OHT and administering auxin that activates the degron element and causes ubiquitin-mediated degradation of the enzyme (bottom part of the schematic).

Figure 4

(A) Schematic representation of ZFNs showing the DNA binding and the FOK1 nuclease domains. Two zinc fingers bind left and right the site of DSB generation and localize the activity of FOK1 in a DNA molecule. The DNA binding domains are frequently designed to recognize a 9 bp target sequence. The FOK1 nuclease cuts the DNA strand 5–7 bp 3′ of the target site. Depending on the design of the target sites, expression of a ZNF nuclease will result in the formation of cohesive or blunt DNA ends. (B) Schematic representation of TALENs showing the DNA binding and the FOK1 nuclease domains. Targeting sites and spacer regions are indicated. Here again, cohesive or blunt ends are generated at the DSB depending on the selection of the recognition sites left and right the DSB.

Figure 5

(A) Schematic representation of the CRISPR/Cas9 system in S. Pyogenes (see text for details). Cas9 generates blunt ends by cutting 3 bp downstream the PAM region of the target DNA molecule. (B) Schematic representation of Cas9 activation with a chimeric gRNA combining crRNA and tracrRNA (see A; see also text for more details). Cas9 nuclease harbors two nuclease domains: HNH and RuvC-like; mutation of one or both of these nuclease domains results in Cas9 enzymes with “nickase” properties, or with null nuclease activity. The latter form of Cas9 can be tethered to other proteins for gain-of-function DNA sequence-specific operations.

Figure 6

(A) Organization of the HPRT gene in the Chinese hamster C. griseous (Cg) near exons 7 and 8. Possible recognition sites of gRNAs allowing the generation of DSBs at different locations within exons and introns are indicated. (B) Exon 3 of the HPRT gene in H. sapiens (Hs). Possible recognition sites for gRNAs allowing the generation of single DSBs or DSB clusters within exons and introns are indicated. (C) Constructs carrying different combinations of I-SceI sites engineered at different distances to model DSB clusters of increasing complexity. The schematic shows I-SceI constructs that would generate upon integration in the genome of a cell, single DSBs, DSB pairs, DSB quadruplets or a cluster of six DSBs. The distances shown are arbitrary and chosen only for illustration purposes.