Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
ReviewEvidence for base excision repair processing of DNA interstrand crosslinks
Introduction
Cellular DNA is under constant threat from endogenous sources such as reactive oxygen species (ROS) and exogenous sources such as environmental oxidants, alkylating agents and anticancer drugs. The most common DNA lesions are base modifications such as alkylation, oxidation, loss of bases and single strand breaks. Complex and more toxic lesions include crosslinks and double strand breaks [1], [2]. Cells are endowed with the inherent capacity to respond to and eliminate these DNA lesions. The lesions are typically recognized and removed by various DNA repair pathways [3]. The base excision repair (BER) pathway as its name suggests is mainly involved in the excision of damaged bases from the DNA. It is considered as the predominant repair system in the protection of cells against a broad range of small base lesions resulting from oxidation, alkylation and deamination [4]. The BER pathway is a highly conserved, multistep process which requires the concerted action of several proteins [5]. It has been estimated that cells encounter ∼10,000 damaged bases per day, most of which are removed by BER [6], [7], [8].
The initiation of BER occurs by the action of DNA glycosylases which recognize alterations to the DNA bases and remove the altered bases by hydrolyzing the N-glycosidic bond. Once the damaged base is removed by a glycosylase, the resulting sugar–phosphate backbone without the base is called an apurinic/apyrimidinic (AP) site [9], [10]. AP endonuclease1 (APE1) cleaves the phosphate backbone resulting in a nick with a 3′ hydroxyl group and 5′deoxyribose phosphate (dRP) residue. The oxidation/reduction state of this 5′deoxyribose is a crucial factor in determining the subsequent downstream processing. If the dRP is not oxidized/reduced, this will lead to the activation of the short-patch BER pathway with the recruitment of DNA Polymerase β (Pol β). The dRP is cleaved by the lyase activity of Pol β and the one nucleotide gap is also filled by Pol β. The final nick is subsequently ligated by the DNA ligase III and XRCC1 complex [10]. If there is any change in the oxidative state of the dRP residue, this leads to the inhibition of the lyase activity of Pol β and activation of other polymerase activity resulting in strand displacement which leads to a 2–10 nucleotide flap intermediate, which is cleaved by FEN1 and joined by DNA Ligase-I [11]. The latter process is termed long-patch repair and requires the action of PCNA [10]. In addition to the oxidized state of the dRP residue, lesion specificity, protein–protein interaction and cell cycle status can also influence the specific choice of BER sub-pathways [12], [13]. The nucleotide incision pathway (NIR) is suggested to be the backup of the BER pathway where Ape1 incises the damaged DNA independent of glycosylase cleavage [14].
Recent studies indicate that BER proteins have broad substrate specificity and they interact with each other to catalyze the repair of DNA lesions [15], [16]. However, in the context of drug therapy, effective BER can render cells resistant to alkylating agents by repairing the DNA adducts that would otherwise be cytotoxic [17], [18]. For example, BER repairs the DNA lesions induced by alkylating agents such as methyl methane sulphonate (MMS) and temozolomide and over expression of BER proteins enhance resistance to these drugs [19], [20]. Therefore, several attempts have been made to target the BER proteins to increase cell sensitivity to alkylating agents [21], [22]. Generation of knock-out mice and identification of small molecule inhibitors of BER proteins have proven to be useful tools to dissect the mechanisms of drug resistance. Several small molecule inhibitors of APE1, Pol β and PARP were tested extensively for their ability to enhance the cytotoxicity of alkylating anticancer agents and some of them have been successful in clinical trials [23], [24], [25], [26], [27], [28]. BER proteins interact with proteins from other DNA repair pathways and this cross-talk/co-ordination has implications for combination therapy targeting two DNA repair pathways simultaneously [29], [30].
Section snippets
Interstrand crosslinks (ICLs)
DNA interstrand crosslinks are formed between both strands of DNA and these covalent links are highly toxic to cells [31], [32]. It has been shown that it takes only a single ICL to kill repair-deficient bacteria and yeast, and about 40 ICLs to kill repair-deficient mammalian cells [33], [34]. The ICLs form an absolute block to metabolic processes such as DNA replication and transcription, trigger cell cycle arrest and apoptosis, ultimately resulting in cell death [35]. In addition, ICLs are
Glycosylases
In BER, specific DNA glycosylases recognize corresponding damaged bases and cleave the N-glycosidic bond between abnormal bases and deoxyribose, leaving either an abasic site or a DNA single-strand break [78]. Several DNA glycosylases are identified in humans, which bind specifically to the modified base initiating the BER pathway. Cell survival assays display differential effects of glycosylases on the sensitivity of ICL inducing agents. 3-alkyladenine-DNA glycosylase (AAG), also called
Endonucleases
Ape1/Ref1 performs complex functions in cells through redox dependent and independent mechanisms. It acts as a transcriptional coactivator, regulates apoptosis, proliferation, differentiation, and production of ROS. In the BER pathway, it acts as an endonuclease that cleaves the abasic sites generated by the action of DNA glycosylases [92], [93]. Over expression of Ape1 has been observed in cancer cells and tumor samples which serves as a diagnostic and prognostic marker and also correlates
Polymerases
Polymerase β (Pol β) belongs to the X family DNA polymerases and is well characterized for a role in DNA repair [125]. Studies have established the role of Pol β in both short-patch and long-patch BER [126]. Pol β has dRP lyase activity which is shown to be a rate-limiting step in the BER pathway [127]. This gap filling polymerase has been identified as being error prone which is evident by increased mutagenesis when it is over expressed. Over-expression of Pol β leads to bifunctional DNA
Other BER proteins
BER proteins including XRCC1 and PARP are also involved in processing ICLs. XRCC1 is involved in the repair of single strand breaks (SSB) generated during BER and acts as a scaffold, connecting other BER proteins such as PARP, Pol β and DNA ligase III [151]. Polymorphisms in the XRCC1 gene have been associated with increased risk of developing certain cancers and also used as a prognostic marker in platinum-treated lung and gastric cancer patients [151], [152], [153]. Down regulation of XRCC1
Conclusion
ICLs covalently link the two strands of DNA and block the denaturing cellular processes that occur during DNA replication and transcription. ICLs are cytotoxic DNA lesions that are formed by a variety of anticancer drugs such as cisplatin, mitomycin C, psoralen, nitrosureas and nitrogen mustard derivatives. These crosslinking agents distort the DNA double helix, each in a unique manner. The repair of ICLs is still not completely understood in eukaryotes and the participation of different DNA
Conflict of interest
None declared
Acknowledgement
We thank members of the Patrick lab for critical reading of the manuscript. This study was supported by a grant from the National Institutes of Health (1R01-GM088249) to SMP.
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