PARP1-MGMT complex underpins pathway crosstalk in O6-methylguanine repair

Abstract

DNA lesions induced by alkylating agents are repaired by two canonical mechanisms, base excision repair dependent on poly(ADP) ribose polymerase 1 (PARP1) and the other mediated by O6-methylguanine (O6meG)-DNA methyltransferase (MGMT) in a single-step catalysis of alkyl-group removal. O6meG is the most cytotoxic and mutagenic lesion among the methyl adducts induced by alkylating agents. Although it can accomplish the dealkylation reaction all by itself as a single protein without associating with other repair proteins, evidence is accumulating that MGMT can form complexes with repair proteins and is highly regulated by a variety of post-translational modifications, such as phosphorylation, ubiquitination, and others. Here, we show that PARP1 and MGMT proteins interact directly in a non-catalytic manner, that MGMT is subject to PARylation by PARP1 after DNA damage, and that the O6meG repair is enhanced upon MGMT PARylation. We provide the first evidence for the direct DNA-independent PARP1-MGMT interaction. Further, PARP1 and MGMT proteins also interact via PARylation of MGMT leading to formation of a novel DNA damage inducible PARP1-MGMT protein complex. This catalytic interaction activates O6meG repair underpinning the functional crosstalk between base excision and MGMT-mediated DNA repair mechanisms. Furthermore, clinically relevant ‘chronic’ temozolomide exposure induced PARylation of MGMT and increased binding of PARP1 and MGMT to chromatin in cells. Thus, we provide the first mechanistic description of physical interaction between PARP1 and MGMT and their functional cooperation through PARylation for activation of O6meG repair. Hence, the PARP1-MGMT protein complex could be targeted for the development of advanced and more effective cancer therapeutics, particularly for cancers sensitive to PARP1 and MGMT inhibition.

Synergistic Antitumor Activity of Talazoparib and Temozolomide in Malignant Rhabdoid Tumors

“…It is likely that the tumor microenvironment and genomic background contribute to the absence of TLZ activity in mice. We recently reported that PARylated MGMT has increased activity in repairing O 6 -methylguanine lesions, which is dependent on alkylating DNA damage [53]. Since resistance to TMZ can be mitigated with PARP1 inhibitors regardless of MGMT expression, mismatch repair proteins may contribute to O 6 -methylguanine repair when MGMT activity is compromised (including through PARP1 inhibition by drugs like TLZ) [77].…”

Exploration of the Use of Natural Compounds in Combination with Chemotherapy Drugs for Tumor Treatment

“…Causing tumor cell death by inducing DNA damage is the main mechanism by which some chemotherapeutic drugs work, while tumor cells rescue themselves from the damage by their own DNA repair function thus creating resistance to chemotherapeutic drugs. O (6)-methylguanine-DNA methyltransferase (MGMT) is an important transferase in the DNA repair process for the elimination of toxic and premutagenic DNA adduct O 6 -methylguanine from cells [ 100 ]. Lipoic acid, disulfide-containing substance, is a naturally occurring cofactor for the mitochondrial enzymes pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase [ 101 ].…”

The DNA Alkyltransferase Family of DNA Repair Proteins: Common Mechanisms, Diverse Functions

“…AGT has also recently been shown to directly interact with poly(ADP-ribose) polymerase 1 (PARP1) [ 121 ]. PARP1 functions in BER as well as other DNA repair pathways by adding poly-ADP-ribose chains (PAR) to its protein targets, as well as itself.…”

“… Posttranslational modifications of AGT. ( A ) Interactions between AGT and PARP1 were demonstrated by co-immunoprecipitation and western blot (WB) analysis after treating cells with temozolomide (TMZ) [ 121 ] ( top ). Immunoglobulin 1 (IgG1) served as a negative control.…”

“…Immunoglobulin 1 (IgG1) served as a negative control. ( Bottom ): SDS PAGE/Western Blot (WB, PAR detection) showed the strongest PARylation of AGT by PARP1 in the presence of O 6 -methylguanine containing dsDNA (dsOligos1 and 3, lanes 9 and 22), as well as PARP1 autoPARylation [ 121 ]. ( B ) ( top ): Active AGT (green) is phosphorylated in vivo by kinases and dephosphorylated by phosphatases, and this reversible phosphorylation inactivates the protein (red).…”