DNA interstrand crosslink repair in mammalian cells.

Interstrand crosslinks (ICLs) are a potent form of DNA damage in which the strands are covalently linked by a bifunctional chemical. The result is a block of normal DNA replication and transcription. The most readily available template for repair, the opposing strand, is also involved with the damage, complicating error-free repair. Straight forward excision and gap filling, as with nucleotide excision repair (NER), seems to be precluded. The many steps involved in IC makes ICL- inducing agents attractive as chemotheraputic drugs (e.g. cisplatin). However, not all ICLs are products of exogenous chemicals; ICLs can also be created by byproducts of metabolism, including the lipid peroxidation product malondialdehyde (Minko et al., 2008). Thus, we find ICL repair mechanisms in organisms from bacteria through humans. In E. coli, the ICL repair pathway utilizes both nucleotide excision repair (NER) and homologous recombination (HR) (Cole, 1973), apparently acting in a single pathway. UvrABC incises the DNA 5′- and 3′- of the ICL on one strand, and the 5′-exonuclease activity of DNA polymerase I creates a single stranded DNA (ssDNA) region required for RecA-mediated recombination. Strand invasion creates a structure on which UvrABC can act, removing the ICL-containing DNA fragment (Dronkert and Kanaar, 2001). The gap is filled by DNA polymerase I and covalently bonded by polynucleotide ligase. In Sacchromyces cerevisiae, studies have shown many genes are involved in ICL repair. Genetic evidence indicates there are three distinct ICL repair pathways in S. cerevisiae, representing ‘NER’, post-replication repair, and HR represented by SNM1, REV3, and RAD51 epistasis groups respectively (Grossmann et al., 2001). Apparently early in ICL repair double strand breaks (DSB) are formed as intermediates (Jachymczyk et al., 1981). Subsequently, a recombination-dependent step utilizing RAD51 may act in completing ICL repair, through repair of the DSB (Jachymczyk et al., 1981). This occurs only when a homologous sequence is available. Post-replication/translesion synthesis utilizing error-prone polymerases such as ζ (REV3/Rev7) or η might replicate past the ICL following DSB formation (Jachymczyk et al., 1981). While rev3 mutants are sensitive to ICL damage, yeast DNA polymerase η mutants show normal sensitivity to ICL, suggesting no role for this bypass polymerase in repair (Grossmann et al., 2001). Less is known about this pathway than NER and HR repair; however, it appears that the pathway allows cells to bypass an ICL as opposed to actually repairing the lesion (Dronkert and Kanaar, 2001). Yeast snm1 Δ mutants are specifically sensitive to ICL (Henriques et al., 1997) but display normal incision (Li and Moses, 2003). They do not, however, resolve the DSB and restore high molecular weight DNA after cross-links (Magana-Schwencke et al., 1982) (Li et al., 2005). Apparently the SNM1 protein, known to be a 5’-exonuclease (Li et al., 2005), acts to modify intermediates of DSB repair. Interestingly, snm1 mutants show normal processing of mating type, indicating that the DSBs occurring in that pathway are processed normally. This leads to the conclusion that the DSBs arising during the ICL repair process have a specific structure, different from DSBs created in mating type switching, thus requiring different processing. As noted, ICL repair appears to involve DSB intermediates; the processing of the DSB resulting from ICL repair requires specific activities peculiar to the process. Therefore, while RAD51, for example, is required for repair of DSBs after ionizing radiation or ICLs, SNM1 is required only for ICL DSB repair. Such a comparison illustrates that some components of ICL repair may act in a general DSB response whereas others act specifically. It appears that the DSBs occurring during normal DNA replication use many of the same components that ICL repair utilizes for genome stability and provide a substrate for HR (Ward et al., 2007). The mammalian ICL repair pathway is more complex than E. coli or yeast. Like yeast, ICL repair operates through a DSB intermediate similar to those occurring in S-phase during replication [reviewed in (Patel and Joenje, 2007)]. While NER and HR pathways are implicated in ICL repair (De Silva et al., 2000; Zheng et al., 2003), other proteins, not found in E. coli or S. cerevisiae are involved. The Fanconi anemia (FA) pathway, as demonstrated by the extreme sensitivity of patients, model organisms, and cell lines to ICLs, is involved in mammalian ICL repair [reviewed in (Kennedy and D’Andrea, 2005; Patel and Joenje, 2007; Wang, 2007)]. Activation of the FA pathway occurs during normal S-phase, likely in response to DSBs or other events during replication, and also occurs with hydroxyurea (HU) treatment which results in stalled replication forks with no ICLs, leading to DSBs (Diffley et al., 2000; Fox, 1985). These observations suggest that DSBs caused by HU (substrate depletion) may be equivalent to those arising from ICL repair. FA patients also exhibit chromosome instability, leading to chromosomal radial formations (Figure 1), levels of which are increased as a result of ICL exposure. In fact, radials are not observed in normal cells without ICL damage at high levels. This implies an overload of the repair system in FA cells. The radial formations may occur as ‘quadriradials’ showing symmetry of four arms, or as adhesions of less than two complete sets of chromatids. The finding of radials is accompanied by apparent breaks in chromatids (Figure 1). Figure 1 Metaphase spread demonstrating chromosome radials. The participation of multiple repair pathways in ICL repair raises the question of whether there is a single monolithic repair response to ICLs in mammals, or if the pathways are independent, as has been unambiguously shown for yeast (Grossmann et al., 2001; McHugh et al., 2001). While models for a unitary path involving NER, HR and post-replication have been suggested (Niedernhofer et al., 2005), results from mutant cell lines and siRNA depletion indicate there are multiple independent pathways in mammals (Hanlon Newell et al., 2008; Hemphill et al., 2008; Wang, 2007) (Jakobs, unpublished). In addition, it appears several bypass DNA polymerases, including Pol κ, may be involved (Minko et al., 2008), not just Pol ζ, further complicating interpretaion.