MLH1‐mediated recruitment of FAN1 to chromatin for the induction of apoptosis triggered by O⁶‐methylguanine

Abstract

O6‐Methylguanines (O6‐meG), which are produced in DNA by the action of alkylating agents, are mutagenic and cytotoxic, and induce apoptosis in a mismatch repair (MMR) protein‐dependent manner. To understand the molecular mechanism of O6‐meG‐induced apoptosis, we performed functional analyses of FANCD2 and FANCI‐associated nuclease 1 (FAN1), which was identified as an interacting partner of MLH1. Immunoprecipitation analyses showed that FAN1 interacted with both MLH1 and MSH2 after treatment with N‐methyl‐N‐nitrosourea (MNU), indicating the formation of a FAN1‐MMR complex. In comparison with control cells, FAN1‐knockdown cells were more resistant to MNU, and the appearances of a sub‐G1 population and caspase‐9 activation were suppressed. FAN1 formed nuclear foci in an MLH1‐dependent manner after MNU treatment, and some were colocalized with both MLH1 foci and single‐stranded DNA (ssDNA) created at damaged sites. Under the same condition, FANCD2 also formed nuclear foci, although it was dispensable for the formation of FAN1 foci and ssDNA. MNU‐induced formation of ssDNA was dramatically suppressed in FAN1‐knockdown cells. We therefore propose that FAN1 is loaded on chromatin through the interaction with MLH1 and produces ssDNA by its exonuclease activity, which contributes to the activation of the DNA damage response followed by the induction of apoptosis triggered by O6‐meG.

FAN1, a DNA Repair Nuclease, as a Modifier of Repeat Expansion Disorders

“…The FAN1-MLH1 interaction may play a structural role, which potentially determines DNA cleavage specificity. Finally, it has been proposed that FAN1 interacts with MSH2 following treatment with alkylating agents [43], but this finding has not been independently confirmed and direct interaction of FAN1 with MSH2 (or its partners MSH3 or MSH6) was not demonstrated.…”

“…Thus, functional MMR is essential to ensure the fidelity of DNA replication but also for genome integrity in non-mitotic cells. Cells treated with the alkylating agent, N-methyl Nnitrosourea (MNU), accumulate FAN1 nuclear foci in an MLH1-dependent manner [43]. Authors of this study propose that FAN1 exo-nuclease generates ssDNA to activate a DNA damage response, ultimately triggering apoptosis [43].…”

“…Cells treated with the alkylating agent, N-methyl Nnitrosourea (MNU), accumulate FAN1 nuclear foci in an MLH1-dependent manner [43]. Authors of this study propose that FAN1 exo-nuclease generates ssDNA to activate a DNA damage response, ultimately triggering apoptosis [43]. To formally prove a role of FAN1 in MMR, one should monitor the rate of G:C-to-A:T transversion mutations in a FAN1-deficient background following MNU treatment.…”

“…Expression of FAN1 is high in brain cells, supporting its possible role in protecting against repeat instability. It is an interacting partner of MMR proteins, notably MLH1 [4,5,7,34,43], suggesting that it may be involved in MMR. Knowing how disruption of the FAN1-MLH1 interaction affects the severity of repeat expansion could be relevant to prediction of the onset of neurological disorders.…”

DNA Mismatch Repair and its Role in Huntington’s Disease

“…These findings suggest a generalized protective role for FAN1 in repeat expansion, although the mechanistic bases remain unclear. Nevertheless, a physical interaction between FAN1 and MLH1 has been documented, and this interaction has been suggested to play a role in the DNA damage response [108,[207][208][209]. Also, it has been suggested that FAN1 binding to CAG extrusions may block access of MutS␤ such structures, thus preventing CAG expansion [158,210].…”