The cytotoxicity of SN1-type alkylating agents such as N-methyl-N'-nitrosourea (MNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or the cancer chemotherapeutics temozolomide, dacarbazine and ...streptozotocin has been ascribed to the persistence of O 6-methylguanine (meG) in genomic DNA. One hypothesis posits that meG toxicity is caused by futile attempts of the mismatch repair (MMR) system to process meG/C or meG/T mispairs arising during replication, while an alternative proposal suggests that the latter lesions activate DNA damage signaling, cell cycle arrest and apoptosis directly. Attempts to elucidate the molecular mechanism of meG-induced cell killing in vivo have been hampered by the fact that the above reagents induce several types of modifications in genomic DNA, which are processed by different repair pathways. In contrast, defined substrates studied in vitro did not undergo replication. We set out to re-examine this phenomenon in replication-competent Xenopus laevis egg extracts, using either phagemid substrates containing a single meG residue, or methylated sperm chromatin. Our findings provide further support for the futile cycling hypothesis.
Replicative DNA polymerases are high fidelity enzymes that misincorporate nucleotides into nascent DNA with a frequency lower than 1/105, and this precision is improved to about 1/107 by their ...proofreading activity. Because this fidelity is insufficient to replicate most genomes without error, nature evolved postreplicative mismatch repair (MMR), which improves the fidelity of DNA replication by up to 3 orders of magnitude through correcting biosynthetic errors that escaped proofreading. MMR must be able to recognize non-Watson-Crick base pairs and excise the misincorporated nucleotides from the nascent DNA strand, which carries by definition the erroneous genetic information. In eukaryotes, MMR is believed to be directed to the nascent strand by preexisting discontinuities such as gaps between Okazaki fragments in the lagging strand or breaks in the leading strand generated by the mismatch-activated endonuclease of the MutL homologs PMS1 in yeast and PMS2 in vertebrates. We recently demonstrated that the eukaryotic MMR machinery can make use also of strand breaks arising during excision of uracils or ribonucleotides from DNA. We now show that intermediates of MutY homolog-dependent excision of adenines mispaired with 8-oxoguanine (GO) also act as MMR initiation sites in extracts of human cells or Xenopus laevis eggs. Unexpectedly, GO/C pairs were not processed in these extracts and failed to affect MMR directionality, but extracts supplemented with exogenous 8-oxoguanine DNA glycosylase (OGG1) did so. Because OGG1-mediated excision of GO might misdirect MMR to the template strand, our findings suggest that OGG1 activity might be inhibited during MMR.
Background: We studied the interplay between base excision repair (BER) of 8-oxoguanine (GO) and mismatch repair (MMR).
Results: BER and MMR interact during the processing of GO/A but not GO/C mispairs.
Conclusion: BER of GO-containing lesions appears to be regulated.
Significance: BER intermediates were believed to be unavailable to other pathways of DNA metabolism. This hypothesis may be incorrect.
The cytotoxicity of SN1-type alkylating agents such as N-methyl-N′-nitrosourea (MNU), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), or the cancer chemotherapeutics temozolomide, dacarbazine and ...streptozotocin has been ascribed to the persistence of O6-methylguanine (meG) in genomic DNA. One hypothesis posits that meG toxicity is caused by futile attempts of the mismatch repair (MMR) system to process meG/C or meG/T mispairs arising during replication, while an alternative proposal suggests that the latter lesions activate DNA damage signaling, cell cycle arrest and apoptosis directly. Attempts to elucidate the molecular mechanism of meG-induced cell killing in vivo have been hampered by the fact that the above reagents induce several types of modifications in genomic DNA, which are processed by different repair pathways. In contrast, defined substrates studied in vitro did not undergo replication. We set out to re-examine this phenomenon in replication-competent Xenopus laevis egg extracts, using either phagemid substrates containing a single meG residue, or methylated sperm chromatin. Our findings provide further support for the futile cycling hypothesis.
PDF