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  • Many DNA repair pathways are candidates for DPC

    2020-04-06

    Many DNA repair pathways are candidates for DPC repair. Nucleotide excision repair (NER) is widely involved in both bacteria and eukaryotes in removing cross-linked protein with low molecular weights (<11kDa) (Nakano et al., 2007, Ide et al., 2011). Homologous recombination (HR) is also involved in some types of DPC (especially >11kDa) (Ide et al., 2011). The predominant pathway chosen by tandospirone may also depend on the type of damage. For example, HR deletion caused the greatest sensitivity under low-dose, chronic exposure to formaldehyde, while NER conferred only low-to-moderate sensitivity under the same exposure. However, following high-dose acute exposure, NER conferred maximal survival, with little contribution of HR (de Graaf et al., 2009). Recently, a process of removing covalently linked proteins by proteolysis in a replication-dependent manner was revealed (Duxin et al., 2014, Stingele et al., 2014). Proteolysis is hypothesized to play a key role in DPC repair by enzymatic digestion. After reducing the size of adducted protein, the bulky protein yields a smaller substrate for canonical DNA repair pathways. Recently, Wss1 has been identified as a member of a protease family for DPC repair in yeast. Stingele et al. argue that DVC1 (DNA damage protein targeting VCP), also called Spartan (SprT-like domain-containing protein), could be a representative ortholog of Wss1 in mammals due to their sequence and motif homology. Primary data confirmed that DVC1 mediates DNA repair by ubiquitinating target proteins independent of HR (Stingele et al., 2014, Stingele et al., 2015). Up to now, the newly found DVC1 with its effect of proteolysis in DPC repair has not been reported. The rapid development of methods for protein labeling and immunodetection provides the possibility to directly detect DPC in living cells with high accuracy and specificity, even to detect a specific protein in the DPC complex (Shoulkamy et al., 2012, Kiianitsa and Maizels, 2013). In this study, harnessing the methodological advance, we focused on NM-induced DNA damage and DPC (mDPC) formation to reveal their characteristics, and then studied the repair manner of DPC. Our results demonstrated that the formation of MGMT-DNA complex in NM-treated cells showed a dose- and time-effect relationship and that proteolysis as well as HR was involved in DPC repair.
    Materials and methods
    Statistical analysis All the sample sizes are at least three. Experiment data were calculated by one-way ANOVA and presented as mean±standard deviation. Bonferroni\'s analysis was used for post-hoc test. P value<0.05 was considered as statistically significant.
    Results
    Discussion In repairing alkylated DNA, the C-terminal domain of MGMT binds to DNA by intercalating a helix residue to the minor groove of DNA. The N-terminal domain, which contains a zinc finger, plays a function through tyrosine residue (Tyr114) bending the DNA chain and flipping out the O6-methylguanine base, which, in turn, allows the active site where Cys145 is located to transfer the alkyl base (Pegg, 2011). However, the situation is different in BAA-induced damage. BAAs can induce mDPC in two ways depending on their specific chemical constitution. As the predominant mechanism, BAAs react with cysteine in MGMT to form a highly reactive half-mustard, leaving one chloroethyl group active. Because of the DNA-binding property of MGMT, the MGMT–HN2 intermediate is likely to access DNA and thus cause a permanent covalent protein adduct, i.e., the MGMT-HN2-DNA cross-links (Liu et al., 2002). The alternative method is that BAAs interact with DNA first and then MGMT (Kalapila et al., 2009). BAAs, such as chlorambucil and HN2, cross-link MGMT to DNA via Cys145 and Cys150. For single guanine conjugation, Cys145 may be a more active site (Loeber et al., 2008). Both of the two ways stabilize the transient interaction between MGMT and DNA during normal alkyl repair, and induced the permanent covalent cross-link. Our hypothesis was that the BAA-mediated anchorage of MGMT to DNA led to the dysfunction of MGMT and the bulky adduct mDPC itself created DNA damage, and as a result, MGMT became a DNA damage enhancer instead of the original important DNA repair enzyme. Our study demonstrated that MGMT was cross-linked with DNA, forming mDPC after HN2 treatment, and the dose- and time-effect relationship were unveiled. In Vlachostergios\' report, tumor cells treated with the alkylating drug temozolomide (TMZ) did not show nuclear MGMT reduction, while proteasome inhibitor bortezomib (BZ) alone or in combination with TMZ caused a significant suppression (Vlachostergios et al., 2013). In our HN2-treated cells, the expression of MGMT was suppressed while the mDPC formation in the nuclear compartment dramatically increased, a situation different from the mono-alkylating agent TMZ.