Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
The mutagenic potential of 8-oxoG/single strand break-containing clusters depends on their relative positions
Highlights
► We determined the mutagenicities of two- and three-lesion clustered damage sites. ► Clustered damage site contains a single strand break (gap) and 8-oxoG(s). ► The mutation frequency of a bi-stranded, but not tandem, two-lesion cluster was high. ► A three-lesion cluster was no more mutagenic than a bi-stranded cluster. ► The relative position of a gap in a cluster is relevant to biological consequences.
Introduction
Ionizing radiation is known to generate various types of DNA lesions, such as base lesions, apurinic/apyrimidinic (AP) sites, and strand breaks. The biological consequences of ionizing radiation are thought to be induced by clustered DNA damage [1], [2], in which two or more lesions are formed within one or two helical turns of the DNA by a single radiation track. Computer simulation studies of interactions of radiation tracks with DNA predict that significant levels of DNA lesions are formed in clusters and that the complexity of the clusters increases with increasing ionization density of the radiation [3], [4], [5]. Sparsely ionizing radiation is predicted to induce additional lesions in close proximity to ∼30–40% of the strand breaks, whereas densely ionizing α-radiation is predicted to induce additional lesions in close proximity to 70–80% of the strand breaks [5]. Clustered DNA damage sites are known to be induced in mammalian cells and E. coli by ionizing radiation, with yields at least four times the yield of double strand breaks (DSBs) [6], [7], [8], [9], [10].
Cells have pathway(s) for preventing mutations from oxidized single lesions. For instance, 8-oxo-7,8-dihydroguanine (8-oxoG) is a well-known mutagenic lesion. To protect against the mutagenic potential of 8-oxoG, E. coli utilizes Fpg which excises 8-oxoG residues from DNA and subsequently cleaves the DNA backbone via its AP lyase activity, leading to a 1-nucleotide gap with 5′- and 3′-phosphate ends [11]. As an additional back-up, MutY excises adenine residues incorporated opposite 8-oxoG after replication [12]. However, when the damage is more complex, as in a clustered damage site, the function of the repair machinery can be compromised [13], [14]. The retardation of repair at sites of clustered DNA damage consisting of closely associated base lesions, such as oxidized purines (8-oxoG) and oxidized pyrimidines (thymine glycol [Tg]), or AP sites has been verified by in vitro analyses using cell extracts or purified proteins [15], [16], [17], [18], [19], [20], [21], [22], [23], [24].
The mutation frequencies (MFs) of bi-stranded clustered damage sites containing a mixture of AP sites, 8-oxoG, dihydrothymine (DHT), Tg lesions in E. coli are reported to be higher than those of isolated single lesions [25], [26], [27], [28], [29], [30]. These studies emphasize the importance of the type of lesions, inter-lesion distance, and relative orientation of lesions for the effective processing of clustered damage sites in the cell. Furthermore, the efficiency or abundance of the specific glycosylase, such as endonuclease III in E. coli, appears to play a decisive role in the initial stages of processing bi-stranded DHT/8-oxoG or Tg/8-oxoG clusters. Endonuclease III removes DHT or Tg initially to give rise to a repair intermediate most likely with an AP site (or a single-strand break (SSB)) opposing 8-oxoG [27], [29]. These in vivo studies and other in vitro studies have demonstrated the existence of a hierarchy of different repair activities in the processing of specific forms of DNA lesion within a clustered damage site, and this interplay determines the outcome of attempted repair at the same time minimizing generation of lethal DSBs. In contrast, clustered damage sites that do not contain base lesions tend to form DSBs during attempted repair, both in vitro and in vivo [15], [20], [31], [32], [33], [34]. Furthermore, addition of AP site(s) or 8-oxoG lesions to a bi-stranded AP sites, which results in a three- or four-lesion cluster, does not prevent the formation of a DSB in vivo [28], [30]. These results suggest that, in most cases, processing of bi-stranded AP clusters is not strongly affected by additional lesions in the cluster, and may lead to the formation of complex DSBs with additional lesions in close proximity to the DSB ends [28], [30].
SSBs are one of the most frequently observed major radiation-induced lesions in cells [35] as well as an intermediate of DNA repair and replication. SSBs are repaired efficiently by the single strand break repair pathway (SSBR), in which each processing step is essentially the same as that of the base excision repair (BER) pathway subsequent to the initial base removal [36]. An unrepaired SSB can act as a strong replicational block in cells [37]. A persistent SSB may lead to a replication-dependent DSB and could be cytotoxic [38]. When a SSB is located opposite an 8-oxoG within a bi-stranded cluster, the repair efficiency of a SSB by cell extracts is about four times lower than that of an AP site placed at the same position [19], [20], [39], [40]. The low repair efficiency of a SSB in a cluster results from inefficient short patch repair of a SSB. These results indicate that a SSB within a clustered damage site is potentially more harmful to cells than an AP site or a base lesion. However, the effect of a SSB associated with base lesions within clustered damage sites on mutagenesis remains largely unknown.
In the present study we have determined the mutagenicity of clustered damage containing a 1-nucleotide gap (GAP), single strand break, located close to an 8-oxoG on the same strand or on the opposing strand. Further, we addressed whether a GAP within a three-lesion cluster enhances the mutagenic potential of 8-oxoG, using wild-type and glycosylase-deficient (fpg, mutY, and fpgmutY) strains of E. coli. The use of this E. coli reporter system has been shown to give similar results when using yeast [33] or a mammalian cell [32] reporter system. We found that some of the GAPs within a cluster play a crucial role in determining the mutagenic potential of the clustered damage site.
Section snippets
Bacterial strains
Isogenic strains CC104 (wild-type) [41], BH540 (fpg::KanR), BH980 (mutY::KanR) were kindly provided by Dr. Serge Boiteux. CSH117 was obtained from the E. coli genetic stock center (CGSC). CC104 fpgmutY (fpg::KanR mutY::TetR) was constructed by P1 transduction of mutY::TetR from CSH117 into BH540 [42].
Preparation of oligonucleotides
Oligonucleotides carrying an 8-oxoG lesion at a fixed position were purchased from Tsukuba Oligo Service (Japan), and two oligonucleotides were used to form strand 1 with a GAP (Table 1). At the
Results
We used a plasmid based assay [26], [27] with E. coli to measure the MF induced by bi-stranded and tandem clustered damage containing a GAP and 8-oxoG(s) within the recognition site of the restriction enzyme Alw26I (Table 1). Ligation mixtures of the plasmid and the damaged DNA was transfected into the wild-type and glycosylase-deficient strains (fpg, mutY and fpgmutY) and propagated in cells. The MF was assessed by the inability of Alw26I to cut the oligonucleotide sequence. The gel assay
Discussion
The preceding results show that the mutagenic potential of 8-oxoG is enhanced when a GAP is closely situated on the opposite strand, but not when placed closely in tandem. Moreover, the MF of a three-lesion cluster consisting of an 8-oxoG and a GAP on one strand and an 8-oxoG on the other strand is no higher than that of a two-lesion bi-stranded GAP/8-oxoG cluster. These results demonstrate that a GAP in a cluster can strongly affect the mutagenic potential of the cluster.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
We thank Drs. K. Fujii and K. Akamatsu for valuable suggestions. We are also grateful to Dr. Q.-M. Zhang-Akiyama for providing us with the P1 phage. This work was in part supported by a Grant-in-Aid for Scientific Research (C) no. 19510062 from the Japan Society for the Promotion of Science (JSPS) and by the Medical Research Council (MRC).
References (47)
- et al.
Reactivity of human apurinic/apyrimidinic endonuclease and Escherichia coli exonuclease III with bistranded abasic sites in DNA
J. Biol. Chem.
(1997) - et al.
In vitro repair of synthetic ionizing radiation-induced multiply damaged DNA sites
J. Mol. Biol.
(1999) - et al.
Clustered DNA damage, influence on damage excision by XRS5 nuclear extracts and Escherichia coli Nth and Fpg proteins
J. Biol. Chem.
(2000) - et al.
8-OxoG retards the activity of the ligase III/XRCC1 complex during the repair of a single-strand break, when present within a clustered DNA damage site
DNA Repair (Amst.)
(2004) - et al.
Repair of tandem base lesions in DNA by human cell extracts generates persisting single-strand breaks
J. Mol. Biol.
(2005) - et al.
An AP site can protect against the mutagenic potential of 8-oxoG when present within a tandem clustered site in E. coli
DNA Repair (Amst.)
(2007) - et al.
Apex1 can cleave complex clustered DNA lesions in cells
DNA Repair (Amst.)
(2009) - et al.
Closely opposed apurinic/apyrimidinic sites are converted to double strand breaks in Escherichia coli even in the absence of exonuclease III, endonuclease IV, nucleotide excision repair and AP lyase cleavage
DNA Repair (Amst.)
(2006) - et al.
Biological consequences of potential repair intermediates of clustered base damage site in Escherichia coli
Mutat. Res.
(2009) DNA damage produced by ionizing radiation in mammalian cells: identities, mechanisms of formation, and reparability
Prog. Nucleic Acid Res. Mol. Biol.
(1988)