Repair of Ionizing Radiation Damage Cellular and Molecular Mechanisms

As mentioned, ionizing radiation can cause a variety of lesions through direct interactions with DNA or, more commonly, through damage induced in adjacent water molecules within a cell or adjacent cells. These damages to DNA include damage to the deoxyribose backbone, base damage, single-strand breaks (SSBs), and double-strand breaks (DSBs).4 Because exposure to ionizing radiation was inevitable during evolution, human cells have developed multiple repair pathways to handle the diverse types of DNA damage created by ionizing radiation.5 Understanding these complex and sometimes redundant repair pathways in human cells has been a major focus in radiation biology during the past 10 to 15 years that will continue in the future.10 These studies on radiation repair pathways also have many links to ionizing radiation effects on the cell cycle, which is discussed later in this chapter.

Repair of Base Damage and DNA Single-Strand Breaks

Repair of ionizing radiation-induced base damage involves a sequence of biochemical processes termed base excision repair (BER). DNA single-strand breaks (SSBs) are one of the most common lesions occurring in human cells, either spontaneously or as intermediates of enzymatic repair of base damage during BER. In BER, a single damaged base or locally multiply damaged bases are recognized and removed by specific glycosylases, resulting in apurinic or apyrimidinic (AP) sites that require cleavage by an AP endonuclease, followed by resynthesis using the complementary strand as a template, and finally by ligation of the repaired strand.11,12 Ionizing radiation-induced DNA SSB repair is completed in steps similar to BER, in which a normal DNA strand serves as a template for repair. When DNA SSBs caused directly by ionizing radiation or arising as BER intermediates are not promptly and efficiently processed, the presence of clusters of damaged sites and of stalled replication forks can then result in DSBs.4

The availability of cellular models characterized by deficiencies in specific DNA repair proteins have proved to be good models to clarify the molecular mechanisms underlying BER and SSB repair.10 For example, it has been shown that transfection of the human gene XRCC1 (X-ray repair cross-complementing gene 1) can correct mouse cells that have a deficiency in rejoining DNA SSB induced by ionizing radiation and alkylating agents. The XRCC1 protein acts as a scaffolding protein, which binds tightly to at least three other factors involved in BER and DNA SSB repair mechanisms including DNA ligase III, DNA polymerase b, and poly (ADP-ribose) polymerase (PARP).12 The importance of XRCC1 in the response of a human cell population to DNA damage has been the subject recently of several studies evaluating whether polymorphism of the human XRCC1 gene contributes significantly to an increased cancer risk in selected popula-tions.12 Indeed, a genetic change in a single amino acid at codon 399 has been linked to an increased risk of several types of gastrointestinal cancers (gastric, pancreatic, colorectal) as well as breast cancer. Additionally, functional analysis of these polymorphisms suggest that these variants of XRCC1 may contribute to a hypersensitivity to ionizing radiation.12

Holliday junction resolution

Repair of DNA Double-Strand Breaks

It is well recognized that the most important lesion caused by ionizing radiation is a DSB.4,5 Unrepaired or misrepaired DSBs can produce chromosomal deletions, translocations, and acentric/dicentric chromosomes, which result in cell lethality or genetic instability. Unlike repair of DNA SSB where the complementary normal DNA strand serves as a template, DSB repair is a more complicated process and can involve homologous recombination (HR) or nonhomologous end joining (NHEJ). Typically, ionizing radiation induces DNA DSBs where one or both DNA ends have a protruding DNA single-strand overhang. It is known from genetic and biochemical studies in radiosensitive yeast mutants that only one unrepaired (or misrepaired) DSB can result in cellular lethality.5,10 The induction of DSBs by ionizing radiation shows a linear function with dose whereas the kinetics of unrepaired (unrejoined) DSBs has a linear quadratic (a, b) relationship with dose.13 With low radiation doses, the quadratic component is insignificant. Thus, the survival curve for a DSB repair-proficient cell would have an initial "shoulder" (a component) at low radiation doses followed by a terminal exponential slope (b component), as previously illustrated in Figure 3.2. In contrast, the "shoulder" region is normally not observed in DSB repair-deficient cells, such as normal skin fibroblasts or normal lymphocytes from patients affected with the autosomal recessive disease ataxia telangiectasia (AT). DSB repair (including both HR and NHEJ) is presumably the fundamental process that mechanistically explains the previously described cellular responses to ionizing radiation damage termed sublethal damage repair (SLDR) and potentially lethal damage repair (PLDR).4 However, in spite of numerous attempts to define DSB repair and survival following ionizing radiation in mathematical terms (such as the LQ model), other contributing factors such as cell-cycle checkpoints, hypoxia, genetic background diversity (polymorphism), induction of apoptosis, and bystander effects (e.g., autocrine or paracrine pathways) may significantly influence the survival response of a cell or tissue (including normal and malignant). These complex genetic and biochemical interactions are not easily simulated by mathematical modeling.

Homologous recombination (HR) is one of two major repair pathways in humans for repair of ionizing radiation-induced DSB. There are three general mechanisms for HR-mediated DSB repair.5,14,15 Two of the HR mechanisms, termed gene conversion and break-induced homology, require homology with a separate DNA molecule (Figure 3.3). The other HR mechanism, single-strand annealing, requires only local homology on one end of the DSB (Figure 3.4). All three

Gene conversion with

Break-induced replication crossing-over figure 3.3. Various pathways of homologous recombination (HR). After 3'-Protruding DNA Single Strand (3'-PSS) are created (arrowheads), they invade a homologous region on another DNA molecule. Replication fork capture (A) results in the formation of a Holliday junction, with its subsequent resolution either with crossing-over (shown) or in the absence of cross-over events. When no replication fork capture occurs (B), the recombination follows the break-induced replication pathway.

HR mechanisms require a 3'-DNA single-strand overhang (3'-PSS). During gene conversion and break-induced replication, 3'-PSSs are created on both ends of a DSB. One then anneals to a homologous region on a sister chromatid, a homologous chromosome, or elsewhere to other chromosomes. New DNA synthesis is next initiated at the 3'-ends and proceeds to the 3'-PSS on the other end of the DSB. At this point, HR can proceed in two different directions, including (a) a Holliday junction (gene conversion), which results from the 3'-PSS annealing to the newly synthesized strand (Figure 3.3A), or (b) the replication fork proceeds until the end of the chromosome without encountering the other end of the DSB (break-induced replication; Figure 3.3B). The third HR mechanism, single-strand annealing, can be synthesis-dependent or


DNA ligase, mismatch repair figure 3.4. Single-strand annealing repairs double-strand breaks (DSBs) that contain both ends having 3'-PSS. Flap endonuclease (FEN-1) removes the misplaced DNA strand.

Strand invasion figure 3.5. Proteins involved in the homologous recombination (HR) pathways of DSB repair: homology search and strand invasion. Taken from combined data obtained on yeast and vertebrate models.

Strand invasion figure 3.5. Proteins involved in the homologous recombination (HR) pathways of DSB repair: homology search and strand invasion. Taken from combined data obtained on yeast and vertebrate models.

-independent (Figure 3.4). Both types of single-strand annealing utilize local homology on the 3'-PSSs of both ends of the DSB. Annealing of the two 3'-PSSs results in a flap of one strand with synthesis-independent annealing or in a gap with synthesis-dependent annealing. The flap is subsequently removed by a 3'^5' exonuclease or a flap endonuclease while a gap is filled by a DNA polymerase.

All three HR mechanisms require the gene products (proteins) of the RAD52 epistasis group (RAD50, 51, 52, 54, 57, 58, and 59) as well as participation of the gene products.15 The Mre11 protein is thought to be a primary sensor of ionizing radiation induced DSB with subsequent recruitment of Rad50 and Xrs2 proteins in yeast and the Nijmegen breakage syndrome (Nbs1) protein in humans (Figure 3.5). The resulting complex of Mre11, Rad50, and Nbs1 is believed to generate 3'-PSS DNA lesions where several homologues of the yeast RAD51 gene (Figure 3.5) next interact with each other in a complex process to facilitate DNA strand migration, invasion, and finally repair.

In contrast, nonhomologous end joining (NHEJ) recombi-national repair does not require extended homology between the ends of a DSB. DSB rejoining can proceed with a limited number of base pairings at the site of the break. In humans, the complex of repair proteins for NHEJ involves Ku70, Ku80, DNA-dependent protein kinase catalytic subunit (DNA PKcs), DNA ligase IV, and X-ray cross-complementation (XRCC) 4. According to a current model (Figure 3.6), the Ku70-Ku80 dimer initially binds to the ends of a DSB, and this dimer acts as a helicase to result in local unwinding at the DSB end.15 DNA PKcs is then recruited near the sites of each end of the DSB followed by the XRCC4/DNA ligase IV complex to repair DSBs created by restriction enzymes. NHEJ can be divided into several pathways, depending on the type of DNA lesion detected. Rejoining of DNA DSB containing four base pair complementary ends created by restriction endonucleases is very efficient and precise. However, when the DSB ends are not complementary, repair is less efficient and may result in small insertions or deletions in the repair of noncomplementary (difficult) DSBs.

It is not clearly understood how human cells choose the pathway for DSB repair.10 Two models have been proposed to explain how a cell might regulate whether HR or NHEJ pathways are used following ionizing radiation-induced DSBs. According to the first model, NHEJ is the major pathway active during the G1 and early S phases of the cell cycle.16 As sister chromatids occur during late S and G2 phases, HR is the major DSB repair pathway at these cell-cycle phases.5 The second model involves a direct competition for DNA DSB ends between the sensors of NHEJ and HR (see Figures 3.5, 3.6). Evidence for the first model is derived from murine scid cells, which lack NHEJ but can repair DSBs during the G2 phase via HR pathways.17 Evidence for the second model is found in human cells where the human Rad52 protein and the Ku70-Ku80 protein complex have been shown to compete to protect DNA DSB ends against exonuclease activity.18 Additionally, the p53 tumor suppressor gene also appears to play a role in a human cell decision between NHEJ and HR following ionizing radiation damage.10 It has been shown that human cells lacking functional p53 (either by null mutations or by mutant p53 expression) display up to 20-fold-higher rates of HR following ionizing radiation damage than cells expressing wild-type p53. Because p53 regulates the transcription and posttranslational activity of RAD51, it may be that the Rad51 protein plays a pivotal role in channeling DSB repair via the NHEJ pathway. Thus, these data suggest that the choice between NHEJ and HR is a function of cell-cycle phase, homologue availability, and the genetic background of a cell (e.g., p53 status).

The consequences of incomplete or faulty DNA repair of ionizing radiation damage may result in carcinogenesis in human normal cells or in the development of ionizing radiation resistance in human tumor cells. A number of genes whose products are involved in DSB repair have been found mutated in many different human cancers. For example, loss of heterozygosity (LOH) of RAD51, RAD52, and RAD54 have been found in human breast carcinomas. Additionally, nearly two-thirds of human pancreas cancers have overexpression of

figure 3.6. A model for nonhomologous end joining (NHEJ) involving DNA-PK. The Ku80/Ku70 complex senses and binds to DSB ends and recruits DNA-PKcs. Ku-associated helicase activity (WRN in the presence of RP-A?) is activated, and the Ku-complex migrates into the double helix with the Ku80 protein heading first in the 5'-direction of the broken end of DNA. It is speculated that, depending on the type of the DSB, either the XRCC4/DNA ligase IV complex or other proteins (i.e., nucleases and recombinases) are recruited to aid in the rejoining of the four broken ends of DNA.

figure 3.6. A model for nonhomologous end joining (NHEJ) involving DNA-PK. The Ku80/Ku70 complex senses and binds to DSB ends and recruits DNA-PKcs. Ku-associated helicase activity (WRN in the presence of RP-A?) is activated, and the Ku-complex migrates into the double helix with the Ku80 protein heading first in the 5'-direction of the broken end of DNA. It is speculated that, depending on the type of the DSB, either the XRCC4/DNA ligase IV complex or other proteins (i.e., nucleases and recombinases) are recruited to aid in the rejoining of the four broken ends of DNA.

the Rad51 protein, which could lead to cellular resistance to ionizing radiation damage and the development of tumor heterogeneity.

There is an ongoing search for new proteins responsible for ionizing radiation-induced DNA damage detection and repair.10,15 During the past decade, several important DNA repair genes were discovered, mutations of which led to defects in DNA repair and extreme sensitivity to ionizing radiation. The X-ray cross-complementing (XRCC) genes were identified in humans and subsequently nine genetic complementation groups were recognized. As mentioned previously, the product of the XRCC1 gene was found to be important for DNA SSB repair. XRCC2 and XRCC3 gene products are part of the RAD51 family and are essential for the HR pathway in DNA DSB repair. XRCC4 to XRCC7 genes initially appeared to be involved in the NHEJ pathway for DNA DSB repair and were later sequenced to reveal that XRCC4 was DNA ligase IV, XRCC5 was Ku80, XRCC6 was Ku70, and XRCC7 was found to be DNA PKcs. Mutants within the XRCC8 complementation group show phenotypic similarities with ataxia telangiectasia (AT) and the Nijmegen breakage syndrome (NBS) with extreme sensitivity to ionizing radiation and to topoisomerase 1 inhibitors. Finally, the XRCC9 gene (also called Fanconi anemia G group) shows marked sensitivity to ionizing radiation and DNA crosslink-ing agents as well as spontaneous chromosome instability.

During the past few years, the RAD24 gene group members were identified to also include RAD9, RAD17, MEC3, and DDC1 genes. The products of this RAD24 epistaxis group appear to have regulatory roles that connect ionizing radiation-induced DNA repair and cell-cycle progression. It is also recognized that products of the tumor suppressor genes such as BRCA1, ATM, and p53 interact with RAD50 and RAD51 gene products to complete the complex process of DNA DSB damage recognition and repair.

Clearly, the field of DNA DSB repair is complex and is an area of intense research for the discipline of radiation oncol-ogy.5,10,15 Although our knowledge of these complex interactions leading to DNA DSB repair is probably still quite rudimentary, translational radiation biologists/oncologists are beginning to explore how some of these genes or protein products might be therapeutic targets for modifying the ionizing radiation response in resistant human cancers. These translational approaches to novel "targeted" therapy in radiation oncology are discussed later in this chapter. The effects on ionizing radiation damage and repair on cell-cycle checkpoints are described in the next subsection.

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