DNA damage that causes a distortion in the shape of the DNA helix such as those caused by intrastrand dimers can be repaired by nucleotide excision repair (NER) [120-122] (Fig. 6.3). There are many polypep-tides involved in this repair system (for review see [18,122]). The DNA lesion is recognized by a polypeptide complex and a 27-29 nucleotide long region around the lesion is removed. The gap is then filled by a complex of DNA replication proteins and the repaired region is ligated to complete the patch [123,124]. NER is also called ''transcription-coupled repair'' because it preferentially repairs actively transcribed DNA strands. This pathway is thought to be responsible for the repair of DNA intrastrand diadducts resulting from cisplatin treatment [18,124,125].
Although alkylation of the O6 position of guanine is thought to be the most important adduct that results from alkylating agents, N7-methylguanine and N3-methyladenine also result from TMZ. These adducts are repaired by the Base Excision Repair Pathway (BER; Reviewed in ; Fig. 6.4). Poly(ADP-ribose) polymerase-1 (PARP-1) is activated by DNA damage and participates in repair by interacting with the BER complex. Inhibitors of PARP-1 have been found to sensitize cells to some DNA methylators [87,126], and inhibitors of PARP have been found to the opened stretch only in the damaged strand, generating a 24-32-base oligonucleotide containing the injury (IV). The regular DNA replication machinery then completes the repair by filling the gap (V). In total, 25 or more proteins participate in NER. In vivo studies indicate that the NER machinery is assembled in a step-wise fashion from individual components at the site of a lesion. After a single repair event (which takes several minutes), the entire complex is disassembled again. (Reprinted from .) See Plate 6.3 in Color Plate Section.
FIGURE 6.4 Base Excision Repair (BER). A battery of glycosylases, each dealing with a relatively narrow, partially overlapping spectrum of lesions, feeds into a core reaction. Glycosylases flip the suspected base out of the helix by DNA backbone compression to accommodate it in an internal cavity of the protein. Inside the protein, the damaged base is cleaved from the sugar-phosphate backbone (stage I in this figure). The resulting abasic site can also occur spontaneously by hydrolysis. The core BER reaction is initiated by strand incision at the abasic site by the APE1 endonuclease (II). Poly(ADP-ribose) polymerase (PARP), which binds to and is activated by DNA strand breaks, and polynucleotide kinase (PNK) may be important when BER is initiated from a SSB to protect and trim the ends for repair synthesis (III). In mammals, the so-called short-patch repair is the dominant mode for the remainder of the reaction. DNA pol b performs a one-nucleotide gap-filling reaction (IV) and removes the 58-terminal baseless sugar residue via its lyase activity (V); this is then followed by sealing of the remaining nick by the XRCC1-ligase3 complex (VI). The XRCC1 scaffold protein interacts with most of the above BER core components and may therefore be instrumental in protein exchange. The long-patch repair mode involves DNA pol b, polS/s and proliferating cell nuclear antigen (PCNA) for repair synthesis (2-10 bases) as well as the FEN1 endonuclease to remove the displaced DNA flap and DNA ligase 1 for sealing (VII-IX). The above BER reaction operates across the genome. However, some BER lesions block transcription, and in this case the problem is dealt with by the TCR pathway described in Fig. 6.3, including TFIIH, XPG (which also stimulates some of the glycosylases) and probably the remainder of the core NER apparatus. (Reprinted from ). See Plate 6.4 in Color Plate Section.
sensitize MMR deficient cells to TMZ . The BER inhibitors AG14361, 3-aminobenzamide, PD128763 and NU1025 have all been shown to sensitize cells to TMZ .
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