Concurrent genetic alterations in DNA polymerase proofreading and mismatch repair in human colorectal cancer

Abstract

Genomic sequences encoding the 3¢ exonuclease (proofreading) domains of both replicative DNA polymerases, pol delta and pol epsilon, were explored simultaneously in human colorectal carcinomas including six established cell lines. Three unequivocal sequence alterations, including one previously reported, were found, and all these were considered as dysfunctional mutations in light of the local amino-acid sequences. In particular, the F367S mutation found in the POLE gene encoding the pol epsilon catalytic subunit, which includes the proofreading domain, is the first found in human diseases. Surprisingly, the tumours carrying these proofreading domain mutations were all defective in DNA mismatch repair (MMR). In addition to the two cell lines with acknowledged MMR gene mutations, the third tumour was also demonstrated to harbour a distinct mutation in MLH1, and indeed exhibited a microsatellite-unstable phenotype. These findings suggest that, in concert with MMR deficiency, defective polymerase proofreading may also contribute to the mutator phenotype observed in human colorectal cancer. Our observations may suggest previously unrecognised complexities in the molecular abnormalities underlying the mutator phenotype in human neoplasms. Keywords: polymerase delta; polymerase epsilon; proofreading domain; colorectal cancer INTRODUCTION Mutation rates on the genome are invariably regulated and typically suppressed to an extremely low level, such as 10 À10 per base replicated, in the organisms. 1 The high fidelity of DNA replication is achieved by several molecular mechanisms. The frequency of erroneous nucleotide incorporation is indeed extremely low compared with that expected from base-pairing energetics, and the vast reduction of the misincorporation frequency involves three steps of molecular events: (a) correct incorporation of complementary nucleotides by DNA polymerases, (b) removal of the newly added nucleotides, particularly incorrectly paired nucleotides, by the 3¢ exonuclease activities associated with the DNA polymerases (proofreading) and (c) postreplicative scanning of mispaired bases by DNA mismatch repair (MMR). 2,3 Various Escherichia coli (E. coli) mutator strains have been used to study these molecular mechanisms by which organisms maintain their mutation rates at very low levels. The dnaQ + (mutD + ) gene of E. coli encodes the epsilon subunit of the DNA polymerase III holoenzyme that is involved in 3¢ exonuclease proofreading activity, 4 and, therefore, the mutation frequencies in the dnaQ mutators are sometimes 1000-10 000 times higher than the wild-type levels. 4,5 In E. coli, MMR is chiefly directed by the proteins encoded by the three genes: mutS + , mutL + and mutH + . 6 The mutS or mutL mutators exhibit an approximately 100 times increase in the mutation frequency. 7 Thus, polymerase proofreading and MMR are the two major

Landscape of somatic single-nucleotide and copy-number mutations in uterine serous carcinoma

“…These tumors with high mutation burden were also notable for having no LOH segments or copy-number variants (CNVs), a feature found in only five other tumors. These features suggest a hypermutator phenotype due to deficiency of mismatch repair (MMR) or polymerase e (POLE) genes (7,8). Consistent with this, these hypermutated tumors showed a paucity of T:A > A:T or C:G > A:T transversions (SI Appendix, Fig.…”

“…This distribution is distinct from the remainder (median 36 protein-altering mutations, all <100). Mutator phenotypes in the absence of germ-line mutations in mismatch repair genes have been seen in several other cancers (7,8). These USC tumors are notable for being relatively frequent and for having a uniformly very high number of mutations, more than those seen in 90% of colon cancers with the mutator phenotype (36).…”

DNA Polymerase ε Deficiency Leading to an Ultramutator Phenotype: A Novel Clinically Relevant Entity

“…In 2010, Yoshida et al reported the first POLE mutation in a human colorectal adenocarcinoma [9]. Since then, multiple germline and somatic mutations in POLD1 and POLE have been reported, predominantly in endometrial cancer and CRC.…”

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants

“…Recently, germline and somatic mutations have been described affecting human POLE, encoding the catalytic subunit of polymerase (Pol) e. Evidence from yeast and human studies indicates that Pol e performs the bulk of leading strand DNA replication (5)(6)(7)(8)(9). POLE mutations affecting the proofreading exonuclease are found in 3% of CRC and 7% of EC (6,(10)(11)(12)(13)(14). Mutations affecting the proofreading function of Pol δ, the main lagging strand DNA polymerase, were also observed, although less frequently (6,(10)(11)(12)(13)(14).…”

“…POLE mutations affecting the proofreading exonuclease are found in 3% of CRC and 7% of EC (6,(10)(11)(12)(13)(14). Mutations affecting the proofreading function of Pol δ, the main lagging strand DNA polymerase, were also observed, although less frequently (6,(10)(11)(12)(13)(14). These observations support the hypothesis that maintenance of DNA replication fidelity restrains neoplasia in humans, as first observed in mice (15)(16)(17), and advance mutator polymerases as important targets to consider for therapeutic intervention.…”