Abstract
Alkylating agents are ubiquitous in our internal and external environments, causing DNA damage that contributes to mutations and cell death that can result in aging, tissue degeneration and cancer. Repair of methylated DNA bases occurs primarily through the base excision repair (BER) pathway, a multi-enzyme pathway initiated by the alkyladenine DNA glycosylase (Aag, also known as Mpg). Previous work demonstrated that mice treated with the alkylating agent methyl methanesulfonate (MMS) undergo cerebellar degeneration in an Aag-dependent manner, whereby increased BER initiation by Aag causes increased tissue damage that is dependent on activation of poly (ADP-ribose) polymerase 1 (Parp1). Here, we dissect the molecular mechanism of cerebellar granule neuron (CGN) sensitivity to MMS using primary ex vivo neuronal cultures. We first established a high-throughput fluorescent imaging method to assess primary neuron sensitivity to treatment with DNA damaging agents. Next, we verified that the alkylation sensitivity of CGNs is an intrinsic phenotype that accurately recapitulates the in vivo dependency of alkylation-induced CGN cell death on Aag and Parp1 activity. Finally, we show that MMS-induced CGN toxicity is independent of all the cellular events that have previously been associated with Parp-mediated toxicity, including mitochondrial depolarization, AIF translocation, calcium fluxes, and NAD+ consumption. We therefore believe that further investigation is needed to adequately describe all varieties of Parp-mediated cell death.
Highlights
DNA alkylation damage, left unrepaired, is mutagenic and cytotoxic, contributing to aging, tissue degeneration and cancer
We optimized a method for the isolation of cerebellar granule neurons (CGNs) from mouse pups to study the effects of alkylation treatment in individual cells cultured ex vivo [22] (Fig 1A)
Initiation of base excision repair (BER) by the Aag DNA glycosylase after methyl methanesulfonate (MMS) treatment induced primary CGN cell death that is mediated through Parp activation
Summary
DNA alkylation damage, left unrepaired, is mutagenic and cytotoxic, contributing to aging, tissue degeneration and cancer. Several different DNA repair mechanisms have evolved to combat these hazardous effects. The base excision repair (BER) pathway repairs two of the most common methylated DNA bases, namely 3-methyladenine (3MeA) and 7-methylguanine (7MeG) [1]. BER of these lesions is initiated by the Aag glycosylase through cleavage of the N-glycosyl bond, producing an abasic (AP) site. The apurinic/apyrimidinic endonuclease 1 (Ape1) cleaves the phosphodiester backbone at the AP site, generating a singlestrand break (SSB) with 3’-OH and 5’-deoxyribosephosphate (5’-dRP) ends. DNA polymerase β (Polβ) removes the 5’-dRP and inserts DNA nucleotides to fill the gap. The SSB is sealed by Ligase I or Xrcc1/Ligase IIIα, completing repair
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