DNA polymerases that produce a faithful copy the genome every time a cell divides have two functional domains: a polymerase domain that takes care of incorporating the correct DNA building blocks (nucleotides), and a proofreading domain that takes care of removing any mis-incorporated and mis-matched nucleotide when this, rarely, occurs.
Sequencing of colorectal cancers and, to a lesser extent, endometrial cancers, has identified a subset of tumors that carry variants in the proofreading domain of the leading-strand replicative DNA polymerase ε (Pol ε), most notably POLE-P286R.
These cancers are marked by a high number of single nucleotide mutations. The origin of mutagenesis in these cancers was initially ascribed to the inactivation of Pol ε proofreading activity, but subsequent work in the yeast S. cerevisiae revealed that the rates of mutation accumulation caused by these Pol ε variants are much higher than would be expected if that were the case (ultra-mutagenesis).
We now provide a detailed study of the mutation frequency, type, sequence context and replication strand bias resulting from a range of DNA polymerase ε variants found in cancers and show that several of them are capable of driving ultra-mutagenesis.
The near identity between the type and frequency of mutations generated by these cancer-associated variants and those generated in proofreading-deficient cells indicate that dysfunctional Pol ε proofreading is responsible for Signature 14, one of the “mutational signatures” identified in human cancers.
Comparison of this mutagenic pattern to that produced by a dysfunctional version of the polymerase domain of Pol ε suggest that the same mutagenic mechanism is active whenever Pol ε is malfunctioning. Mutational patterns specific for proofreading domain variants, however, indicate that these cancer-associated variants introduce additional, characteristic insertions and T>A mutations because they cannot accommodate the mismatched primer in the proofreading domain.
These data provide further insights into how cells normally guard against mutagenesis during DNA replication and how specific point mutations alter DNA polymerase activities. Given the extremely high conservation of molecular mechanisms of DNA replication across all organisms, including humans, these mechanisms are likely to operate in the cancer cells carrying Pol ε proofreading domain alleles.
Highlights from the paper
- Four cancer associated DNA polymerase ε alleles with mutations in the proofreading domain show a range of mutator phenotypes including ultra-mutagenesis, a higher mutation rate than would be expected by loss of proofreading.
- Combination with mismatch repair deficiency shows a synergetic effect leading to dramatically high mutation rates. Analysis of mutation frequencies before mismatch repair correction revealed striking similarities to cancer signature 14.
- Malfunction of polymerase or exonuclease domains of DNA polymerase ε results in similar mutational spectra and strand bias indicating a shared mutagenic mechanism. Mutations that are uniquely introduced by exonuclease domain alleles suggest that these variants of DNA polymerase ε introduce errors by “dislocation” mutagenesis.
Read the article here