**1. Introduction**

*Cis*-diamminedichloridoplatinum(II) (best known as cisplatin) and its derivatives, such as carboplatin and oxaliplatin, are platinum (Pt)-based chemotherapeutics [1]. Since its approval by the FDA (Food and Drug Administration) in 1978, cisplatin has been widely used to treat a number of cancers, including testicular, ovarian, bladder, lung cancer, and others [2]. Cisplatin exerts its anti-cancer activity by inducing cytotoxic DNA lesions after activation in cells by a series of aquation reactions [1]. The aquated cisplatin is highly reactive and prone to interact with a wide range of cellular substrates, including DNA. The activated cisplatin preferentially binds to two purines on the same DNA strand, causing 1,2-intrastrand crosslinks of purine bases such as Pt-d(GpG) and to a lesser extent, Pt-d(ApG) adducts [3]. These two adducts represent about 90% and 10% of cisplatin lesions, respectively [1]. Additionally, cisplatin also binds guanines on the two opposing DNA strands, inducing the formation of interstrand crosslink G-Pt-G. However, the frequency of interstrand lesion is much lower than intrastrand crosslinks. Cisplatin-induced DNA crosslinks can strongly inhibit replicative DNA polymerases and induce apoptosis [4], which explains why cisplatin selectively kills fast-proliferating cancer cells. Additionally, cisplatin adducts block elongating RNA polymerases and inhibit gene transcription [5], which also contributes to cisplatin-induced cell death (Figure 1).

(Figure 1).

**Figure 1.** Cytotoxicity of cisplatin. Cisplatin-induced DNA adduct (red) blocks replicative DNA polymerase (**left**) and RNA polymerase (**right**). Both replication and transcription stalling can trigger apoptosis. **Figure 1.** Cytotoxicity of cisplatin. Cisplatin-induced DNA adduct (red) blocks replicative DNA polymerase (**left**) and RNA polymerase (**right**). Both replication and transcription stalling can trigger apoptosis.

The most impressive clinical success of cisplatin is the cure of over 80% patients with metastatic testicular germ cell tumors (TGCT) [6], a phenomenon that has been attributed to the intrinsic defects in DNA repair in TGCT cells [7,8]. However, the same clinical benefits are not observed for other solid tumors. Some tumor cells, including colorectal, lung, and prostate cancer cells, are intrinsically resistant to cisplatin [9]. Other tumors are originally sensitive to cisplatin, but develop resistance during the process of treatment, when tumor cells acquire further mutations to adapt to cisplatin damage. The acquired cisplatin resistance is particularly common for advanced ovarian cancer patients [10], and there is an urgent need to develop new strategies to reverse drug resistance. Resistance to platinum drugs such as cisplatin is associated with a number of cellular mechanisms, including low drug uptake through reduced expression of the copper transporter protein Ctr1 [11], inhibition of cisplatin potency by proteins such as glutathione (GSH) [12], replicative bypass of cisplatin-induced DNA damage by translesion synthesis DNA polymerases [13], and removal of cisplatin adducts by DNA repair to abolish cytotoxicity [14]. The most impressive clinical success of cisplatin is the cure of over 80% patients with metastatic testicular germ cell tumors (TGCT) [6], a phenomenon that has been attributed to the intrinsic defects in DNA repair in TGCT cells [7,8]. However, the same clinical benefits are not observed for other solid tumors. Some tumor cells, including colorectal, lung, and prostate cancer cells, are intrinsically resistant to cisplatin [9]. Other tumors are originally sensitive to cisplatin, but develop resistance during the process of treatment, when tumor cells acquire further mutations to adapt to cisplatin damage. The acquired cisplatin resistance is particularly common for advanced ovarian cancer patients [10], and there is an urgent need to develop new strategies to reverse drug resistance. Resistance to platinum drugs such as cisplatin is associated with a number of cellular mechanisms, including low drug uptake through reduced expression of the copper transporter protein Ctr1 [11], inhibition of cisplatin potency by proteins such as glutathione (GSH) [12], replicative bypass of cisplatin-induced DNA damage by translesion synthesis DNA polymerases [13], and removal of cisplatin adducts by DNA repair to abolish cytotoxicity [14].

Nucleotide excision repair (NER) plays a critical role in removing helix-distorting DNA damage, including cisplatin-induced intrastrand crosslinks [15–17]. Previous studies in testicular tumors (i.e., TGCT) have shown low NER activity in TGCT cell extracts and low expression of several key NER proteins [7,18]. The intrinsic NER defects in TGCT cells are correlated with high cisplatin sensitivity and high cure rate among TGCT patients [19]. The impressive findings in TGCT triggered investigations in other solid tumors such as lung and ovarian cancer, in order to understand how NER contributes to cisplatin resistance in these tumors. Through extensive studies, it is now evident that elevated NER capacity in cancer cells is generally correlated with drug resistance [20,21]. The expression levels of several NER proteins (e.g., XPC, XPA, ERCC1-XPF, and XPG) and somatic mutations in XPD significantly affect cellular sensitivity to cisplatin [22–24]. Recent CRISPR-Cas9 screening also identified transcription-coupled NER (TC-NER) factors in resolving cisplatin-induced transcription stalling to avoid apoptosis [25]. Hence, the status of these critical NER factors represents a prospective biomarker to predict the outcome of cisplatin treatment. Furthermore, manipulation of cellular NER capacity with specific inhibitors is a potentially useful strategy to cope with cisplatin Nucleotide excision repair (NER) plays a critical role in removing helix-distorting DNA damage, including cisplatin-induced intrastrand crosslinks [15–17]. Previous studies in testicular tumors (i.e., TGCT) have shown low NER activity in TGCT cell extracts and low expression of several key NER proteins [7,18]. The intrinsic NER defects in TGCT cells are correlated with high cisplatin sensitivity and high cure rate among TGCT patients [19]. The impressive findings in TGCT triggered investigations in other solid tumors such as lung and ovarian cancer, in order to understand how NER contributes to cisplatin resistance in these tumors. Through extensive studies, it is now evident that elevated NER capacity in cancer cells is generally correlated with drug resistance [20,21]. The expression levels of several NER proteins (e.g., XPC, XPA, ERCC1-XPF, and XPG) and somatic mutations in XPD significantly affect cellular sensitivity to cisplatin [22–24]. Recent CRISPR-Cas9 screening also identified transcription-coupled NER (TC-NER) factors in resolving cisplatin-induced transcription stalling to avoid apoptosis [25]. Hence, the status of these critical NER factors represents a prospective biomarker to predict the outcome of cisplatin treatment. Furthermore, manipulation of cellular NER capacity with specific inhibitors is a potentially useful strategy to cope with cisplatin resistance.

#### resistance. **2. Mechanism of NER in the Repair of Cisplatin Adducts**

NER targets a wide range of mutagenic and cytotoxic DNA lesions. Common substrates for NER include ultraviolet (UV)-induced pyrimidine dimers, DNA adducts formed by Benzo[a]pyrene (BaP) in cigarette smoke, intrastrand crosslinks, and other helix-distorting DNA lesions [26]. The structural alteration caused by these bulky lesions interferes with DNA replication and gene transcription, and thus, needs to be repaired correctly before leading to permanent mutations or cell death. DNA helix-distorting lesions can be recognized by NER surveillance proteins such as Xeroderma pigmentosum complementation group C (XPC) and the UV-damaged DNA binding protein complex

(UV-DDB) [26]. Once bound to damaged DNA, XPC recruits transcription factor II H (TFIIH), a 10-subunit protein complex consisting of two important DNA helicases, XPB and XPD. XPB and XPD, together with XPA and replication protein A (RPA), separate the two DNA strands around the damage site, creating a pre-incision DNA bubble that is recognized by repair endonucleases ERCC1-XPF and XPG. XPF and XPG cleave the damaged DNA strand on the 50 and 30 side relative to the damage, releasing an oligonucleotide of ~30 nucleotide (nt) containing the lesion (Figure 2). The resulting gap is filled with the activity of DNA polymerase using the undamaged strand as the synthesis template. Finally, DNA ligase I or ligase IIIα is recruited to seal the DNA backbone [26]. As damage recognition by XPC and UV-DDB can occur across the whole genome, this NER subpathway is known as global genomic NER (GG-NER). GG-NER is responsible for the removal for the majority of bulky lesions residing in the genome. NER can also be activated through the stalling of RNA polymerases. When the elongating RNA Pol II is blocked by DNA damage, the prolonged arrest of Pol II serves as a strong signal to activate transcription-coupled NER (TC-NER) [27]. TC-NER is a highly efficient repair mechanism that specifically removes transcription-impeding damage from the transcribed strand (TS) to allow transcription resumption. XPC and UV-DDB are not required for TC-NER. Instead, the stalled RNA Pol II recruits the Cockayne syndrome group B (CSB) protein, a master regulator of TC-NER [28]. CSB coordinates assembly of the downstream NER core factors for dual incision and repair synthesis. Mechanistically, GG-NER and TC-NER mainly differ in damage recognition, but share the same set of core NER enzymes for the "cut-and-patch"-type repair process (Figure 2). complementation group C (XPC) and the UV-damaged DNA binding protein complex (UV-DDB) [26]. Once bound to damaged DNA, XPC recruits transcription factor II H (TFIIH), a 10-subunit protein complex consisting of two important DNA helicases, XPB and XPD. XPB and XPD, together with XPA and replication protein A (RPA), separate the two DNA strands around the damage site, creating a pre-incision DNA bubble that is recognized by repair endonucleases ERCC1-XPF and XPG. XPF and XPG cleave the damaged DNA strand on the 5′ and 3′ side relative to the damage, releasing an oligonucleotide of ~30 nucleotide (nt) containing the lesion (Figure 2). The resulting gap is filled with the activity of DNA polymerase using the undamaged strand as the synthesis template. Finally, DNA ligase I or ligase IIIα is recruited to seal the DNA backbone [26]. As damage recognition by XPC and UV-DDB can occur across the whole genome, this NER subpathway is known as global genomic NER (GG-NER). GG-NER is responsible for the removal for the majority of bulky lesions residing in the genome. NER can also be activated through the stalling of RNA polymerases. When the elongating RNA Pol II is blocked by DNA damage, the prolonged arrest of Pol II serves as a strong signal to activate transcription-coupled NER (TC-NER) [27]. TC-NER is a highly efficient repair mechanism that specifically removes transcription-impeding damage from the transcribed strand (TS) to allow transcription resumption. XPC and UV-DDB are not required for TC-NER. Instead, the stalled RNA Pol II recruits the Cockayne syndrome group B (CSB) protein, a master regulator of TC-NER [28]. CSB coordinates assembly of the downstream NER core factors for dual incision and repair synthesis. Mechanistically, GG-NER and TC-NER mainly differ in damage recognition, but share the same set of core NER enzymes for the "cut-and-patch"-type repair process (Figure 2).

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 3 of 14

distorting lesions can be recognized by NER surveillance proteins such as Xeroderma pigmentosum

NER targets a wide range of mutagenic and cytotoxic DNA lesions. Common substrates for NER include ultraviolet (UV)-induced pyrimidine dimers, DNA adducts formed by Benzo[a]pyrene (BaP) in cigarette smoke, intrastrand crosslinks, and other helix-distorting DNA lesions [26]. The structural alteration caused by these bulky lesions interferes with DNA replication and gene transcription, and

**2. Mechanism of NER in the Repair of Cisplatin Adducts** 

**Figure 2.** Repair of cisplatin-induced DNA damage by NER. Cisplatin damage can be recognized by surveillance protein XPC or elongating RNA Pol II, to initiate GG-NER or TC-NER. After damage recognition, the two subpathways utilize the same set of repair enzymes to conduct DNA unwinding, dual incision, and repair synthesis and ligation. **Figure 2.** Repair of cisplatin-induced DNA damage by NER. Cisplatin damage can be recognized by surveillance protein XPC or elongating RNA Pol II, to initiate GG-NER or TC-NER. After damage recognition, the two subpathways utilize the same set of repair enzymes to conduct DNA unwinding, dual incision, and repair synthesis and ligation.

Previous studies using mammalian cell extracts as well as reconstituted NER system with purified proteins have shown high activity of NER toward cisplatin-induced intrastrand crosslinks [15,29]. These in vitro cisplatin repair experiments used chemically synthesized oligonucleotides containing site-specific cisplatin adducts. Incubation of the DNA substrates with whole cell extracts or purified NER enzymes resulted in 25–29 nt excision products, due to the dual incision by XPF and XPG [15,29]. Using these well-defined NER systems, the abundance of the excision products can be detected to

investigate NER activity for different types of cisplatin adducts. For example, experiments conducted in HeLa cell extracts have shown that NER enzymes exhibit slightly higher repair activity for the intrastrand crosslink Pt-d(ApG) relative to Pt-d(GpG), suggesting the local DNA sequence for the crosslink can affect repair efficiency [15]. Additionally, intrastrand adducts induced by cisplatin and its derivatives, such as oxaliplatin and JM216, can all be repaired by NER proteins with similar repair kinetics [16]. In contrast, the interstrand crosslink G-Pt-G cannot be repaired by NER [15], consistent with findings showing that interstrand crosslinks are repaired by the Fanconi anemia (FA) pathway [30]. Furthermore, published data also uncovered the regulation of cisplatin damage repair by non-NER proteins. In this regard, in vitro studies showed that the high mobility group (HMG)-domain proteins suppress excision repair of the main cisplatin adduct, Pt-d(GpG) [15,29]. HMG proteins bind to cisplatin-damaged DNA by recognizing the altered DNA structure [31], and thus, shield cisplatin adducts from NER [32]. As a result, cisplatin may cause more cytotoxicity in cancer cells with high expression of HMG proteins [33].

Repair of cisplatin damage by NER is also confirmed by experiments conducted in cells. Compared to biochemical analysis, cellular repair studies provided the advantage of analyzing both GG-NER and TC-NER in resolving cisplatin-induced DNA damage. Particularly, the development of high-throughput damage sequencing methods such as Damage-seq and eXcision Repair-seq (XR-seq) has significantly improved the understanding of cisplatin damage distribution and repair kinetics at the genome scale [17,34]. Damage-seq was built on the observation that cisplatin damage blocks replicative DNA polymerases. This method first uses a cisplatin damage-specific antibody to enrich DNA fragments containing cisplatin adducts. The damage-containing single stranded DNA is used as the template to synthesize a new strand with a replicative DNA polymerase, so that the newly synthesized strand terminates at the site of cisplatin damage. By mapping the location of the 30 end of the nascent strand, this method allows for identification of the damage site [17]. On the other hand, XR-seq is used for high-resolution mapping of NER activity in cisplatin-treated cells. In XR-seq, the ~30-nt single-stranded excision repair fragments generated during NER are purified from cells and ligated with sequencing adaptors for next-generation sequencing [34]. By mapping the sequencing reads to the human genome and counting the number of XR-seq reads in different genomic regions, this method offers a genome-wide repair profile for cisplatin adducts as well as other bulky lesions [17]. The XR-seq data have revealed highly variable repair efficiency for cisplatin damage across the genome. Several factors, including transcription and chromatin states, can affect the repair efficiency. Particularly, the transcribed strand of active genes display robust repair by TC-NER at the early repair time points, whereas cisplatin damage located in heterochromatin is poorly repaired [17], likely due to the low access of cisplatin damage to GG-NER proteins. In contrast, Damage-seq data revealed that formation of cisplatin damage is largely uniform across the genome and mainly dictated by the underlying DNA sequences [17]. Thus, these new methods provide high-resolution maps of cisplatin damage formation and repair kinetics at the genome level, and highlight the important roles for both GG-NER and TC-NER in resolving cytotoxic damage induced by cisplatin. Indeed, as discussed below, both NER subpathways have a significant impact on the cisplatin response in cancer cells.
