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| Background: Formation and Repair of DNA Damage How to setup models to simulate the formation and repair of DNA damage |
DNA Damage Damage to a cells DNA is widely viewed as one of the most critical targets of ionizing radiation. Every day massive amounts of DNA damage are produced as the byproducts of a cells normal oxidative metabolism. In a mammalian cell, the number of endogenously produced DNA damage sites is estimated at one million per day (Holmquist 1998) and include damages such as oxidized bases, abasic sites, and strand breaks. The types of damage produced by ionizing radiation are similar to those formed by endogenous processes (Wallace 1998). However, the spatial arrangement of the damage sites in the DNA is quite different. Endogenous processes mainly produce singly damaged sites (SDS), i.e., isolated damages that are separated from each other by many undamaged base pairs, whereas ionizing radiation tends to create clusters of damages within one or two helical turns of the DNA. Such clusters are often referred to as multiply damaged sites (MDS) (Ward 1988). Many low-LET radiations create approximately 1,000 single-strand breaks (SSBs) and 40 double-strand break (DSB) per Gy in a typical mammalian cell (Ward 1988). A DSB is created when a MDS composed of 2 or more strand breaks effectively cuts a DNA molecule into smaller fragments. That is, base pairing and chromatin structure interactions near the strand breaks are unable to keep the damage DNA ends in close spatial proximity to each other. All other singly and multiply damaged DNA sites that are composed of at least one strand break are called a single strand break (SSB). Double strand breaks are widely accepted as the main type of DNA damage responsible for radiation-induced cell killing, and they are also highly mutagenic. However, a variety of experiments have shown that radiation creates many different kinds of MDS in addition to the DSB. Studies show that DSBs comprise only 2030% of the total yield of MDS (Jenner et al. 2001, Sutherland et al. 2000a, Sutherland et al. 2000b, Sutherland et al. 2002), and MDS other than the DSB may make a substantial contribution to radiation-induced mutagenesis and cell killing (Ward et al. 1985, Ward 1995). The underlying hypothesis is that MDS are hard to repair because repair enzymes may not always have an intact template to guide the replacement of damaged nucleotides (Ward 1988, Goodhead 1994). Repair of SDS and MDS other than the DSB The majority of singly damaged sites, ranging from oxidized bases to abasic sites to strand breaks, are repaired by base excision repair (BER) (S.S. Wallace 1994, 1998). Two modes of the BER process have been observed in both prokaryotes (Dianov and Lindahl 1994) and eukaryotes (Matsumoto et al. 1994, Frosina et al. 1996, Klungland and Lindahl 1997). The excision and resynthesis of a single nucleotide, termed short-patch base excision repair (SP BER), occurs in most cases (Nilsen and Krokan 2001). The other mode, long-patch base excision repair (LP BER), results in the removal of fragments 210 nucleotides long. BER is also involved in the repair of MDS other than the DSB (Wallace 1998, Weinfeld et al. 2001). Nucleotide excision repair (NER) is another, enzymatically distinct, DNA repair pathway. In eukaryotes, oligonucleotide fragments approximately 2432 nucleotides in length are replaced by the NER process (Sancar 1996, Wood 1997). NER is the major pathway for the repair of bulky, helix-distorting lesions usually associated with UV light-induced damage. However, oxidative DNA damages that are normally considered substrates for BER can also be recognized and repaired by bacterial (Lin and Sancar 1989, Kow et al. 1990), yeast (Scott et al. 1999, Torres-Ramos et al. 2000), and human (Huang et al. 1994, Reardon et al. 1997) NER pathways. NER is the primary pathway for the removal of reactive oxygen species-induced cyclopurines in mammalian cells (Brooks et al. 2000, Kuraoka et al. 2000). DSB Repair Double strand breaks cause severe disruptions in the structure of the DNA double helix, and DSB repair is essential for cell survival and maintenance of genome. Experiments and theory suggest a causal link between the DSB formation and the induction of mutations and chromosome aberrations (Hlatky et al. 2002, Iliakis et al. 2004, Jackson 2002, Pfeiffer et al. 2004, Sachs et al. 1997). Point mutations and most chromosome aberrations are formed when DSBs are mis-repaired. DSBs, if unrepaired, are usually considered lethal. However, DSBs formed in the latter stages of the S phase and in the G2 and M stages of the cell cycle may not be lethal if the damaged chromosome has an undamaged sister chromatid. Several, evolutionarily conserved DSB repair mechanisms exist in eukaryotes: (1) homologous recombination (HR), (2) non-homologous end joining (NHEJ) and (3) single-strand annealing (SSA). The relative contribution each of these pathways makes to DSB repair depends on the organism, cell type and the cell cycle stage. In yeast, most DSBs are repaired by HR while in the higher eukaryotes NHEJ tends to be more important (Jackson 2002). However in mammals, defects in both HR and NHEJ results in a predisposition towards cancer and, at the cellular level, the frequency of chromosomal aberrations is increased (Pastink et al. 2001). NHEJ predominates in G0 (quiescent cells) and in G1/early S phase cells whereas HR is mainly important in late S/G2 phase when the sister chromatid is available to act as a template for repair (Jackson 2002). NHEJ of DSBs formed by a site-specific endonuclease (I-SceI) usually result in small deletions 1 to 20 or 30 base pair (bp) in length (Liang et al. 1998, Richardson and Jasin 2000). On rare occasions, deletions as large as 299 bp are observed as are insertions ranging in size from 45 to 205 bp (Liang et al. 1998, Richardson and Jasin 2000). DSBs formed by a site-specific endonuclease are highly mutagenic (small probability of correct repair). Radiation-induced DSBs are frequently accompanied by additional strand breaks or base damage (Nikjoo et al. 2001), and the repair of radiation-induced DSBs by NHEJ is most likely as error-prone (or more error-prone) than the repair of DSBs formed by an endonuclease. The rate of DSB rejoining has also been shown to depend on radiation quality and local damage complexity (Pastwa et al. 2003). In contrast to NHEJ, DSB repair by HR is usually accurate and non-mutagenic (Jackson 2002). For HR to occur, a second DNA molecule (or another region of the same DNA molecule) with sequence homology to the region containing the DSB must be available to serve as a template. Although mammalian cells may use homologous sequences located throughout the genome to repair DSBs, the HR process has a strong template bias for the sister chromatid. The sister chromatid is preferred by 2 or 3 orders of magnitude over a homologous or heterologous chromosome (reviewed in Johnson and Jasin 2001). The template bias observed in mammalian cells may reduce the mutagenic potential of the DSB repair process by helping to prevent inappropriate recombination. References
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