Rapid induction of chromatin-associated DNA mismatch repair proteins after MNNG treatment

Abstract

Treatment with low concentrations of monofunctional alkylating agents induces a G 2 arrest only after the second round of DNA synthesis in mammalian cells and requires a proficient mismatch repair (MMR) pathway. Here, we have investigated rapid alkylation-induced recruitment of DNA repair proteins to chromosomal DNA within synchronized populations of MMR proficient cells (HeLa MR) after N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) treatment. Within the first hour, the concentrations of MutSα and PCNA increase well beyond their constitutive chromosomally bound levels and MutLα is newly recruited to the chromatin-bound MutSα. Remarkably, immunoprecipitation experiments demonstrate rapid association of these proteins on the alkylation-damaged chromatin, even when DNA replication is completely blocked. The extent of association of PCNA and MMR proteins on the chromatin is dependent upon the concentration of MNNG and on the specific type of replication block. A subpopulation of the MutSα-associated PCNA also becomes monoubiquitinated, a known requirement for PCNA to interact with translesion synthesis (TLS) polymerases. In addition, chromatin-bound SMC1 and NBS1 proteins, associated with DNA double-strand-breaks (DSBs), become phosphorylated within 1–2 h of exposure to MNNG. However, these activated proteins are not co-localized on the chromatin with MutSα in response to MNNG exposure. PCNA, MutSα/MutLα and activated SMC1/NBS1 remain chromatin-bound for at least 6–8 h after alkylation damage. Thus, cells that are exposed to low levels of alkylation treatment undergo rapid recruitment to and/or activation of key proteins already on the chromatin without the requirement for DNA replication, apparently via different DNA-damage signaling pathways.