Human cells bearing homozygous mutations in the DNA mismatch repair genes hMLH1 or hMSH2 are fully proficient in transcription-coupled nucleotide excision repair

Abstract

The transcription-coupled nucleotide excision repair (TCNER) pathway maintains genomic stability by rapidly eliminating helix-distorting DNA adducts, such as UV-induced cyclobutane pyrimidine dimers (CPDs), specifically from the transcribed strands of active genes. DNA mismatch repair (MMR) constitutes yet another critical antimutagenic pathway that removes mispaired bases generated during semiconservative replication. It was previously reported that the human colon adenocarcinoma strains HCT116 and LoVo (bearing homozygous mutations in the MMR genes hMLH1 and hMSH2, respectively), besides manifesting hallmark phenotypes associated with defective DNA mismatch correction, are also completely deficient in TCNER of UV-induced CPDs. This revealed a direct mechanistic link between MMR and TCNER in human cells, although subsequent studies have either supported, or argued against, the validity of this important notion. Here, the ligation-mediated polymerase chain reaction was used to show at nucleotide resolution that MMRdeficient HCT116 and LoVo retain the ability to excise UV-induced CPDs much more rapidly from the transcribed vs the nontranscribed strands of active genes. Moreover, relative to DNA repair-proficient counterparts, MMR-deficient cells were not more sensitive to the cytotoxic effects of UV, and displayed equal ability to recover mRNA synthesis following UV challenge. These results conclusively demonstrate that hMLH1-and hMSH2-deficient human colon adenocarcinoma cells are fully proficient in TCNER.

Detection of an Involvement of the Human Mismatch Repair Genes hMLH1 and hMSH2 in Nucleotide Excision Repair Is Dependent on UVC Fluence to Cells

“…3). This is consistent with the results of Rochette et al (9), showing a similar removal of CPDs from the transcribed strand of the c-jun and/or p53 gene in the MMR-deficient compared with the MMR-proficient cell lines after a UVC exposure of 15 J/m 2 to these same cell lines.…”

“…Using a strandspecific Southern blot-based assay, Mellon et al (8) reported that the MMR-deficient HCT116 and LoVo human adenocarcinoma cells (carrying mutations in the hMLH1 and hMSH2 genes, respectively) were defective in TCR of CPDs at the active dihydrofolate reductase gene locus. In contrast, using a ligation-mediated PCR that measures CPD removal at nucleotide resolution, Rochette et al (9) have reported recently that these same MMR-deficient adenocarcinoma cells are fully proficient in TCR. In addition, Sonneveld et al (10) reported that hMSH2-deficient murine embryonic fibroblasts are able to remove CPDs much more rapidly from the transcribed compared with the nontranscribed strand of active genes suggesting proficient TCR in murine embryonic fibroblasts, and Adimoolam et al (11) reported that hMLH1-deficient HCT116 cells are fully competent in TCR at the DHFR locus.…”

UV wavelength-dependent regulation of transcription-coupled nucleotide excision repair in p53-deficient human cells

“…We reasoned that this methodological difference is unlikely to account for the observed contrariety in experimental outcome, because each of the aforementioned assays has been rigorously validated as a means to evaluate NER rates along the TS and NTS of active genes in living cells. For example, by using LMPCR, we previously reproduced the expected result, initially obtained via the Southern blot-based method (8), that CS-B cells are deficient in TCNER but not GNER of UV-induced CPD (31). Finally, we note that each of the above-mentioned assays quantifies precisely the same event, i.e., the lesion recognition͞incision step of NER as manifested by the disappearance of T4 endonuclease Vsensitive sites.…”

SW480, a p53 Double-mutant Cell Line Retains Proficiency for Some p53 Functions

“…10 In a previous study, we compared repair of UVinduced CPD in the SW480 cell line and the LoVo cell line, derived from a colorectal adenocarcinoma. 11 The SW480 cell line was derived from a Duke's colon adenocarcinoma that was removed from a 50-year-old male patient. 12 The cell line was submitted to the American Type Culture Collection (ATCC) at passage 91 by Leibovitz in November 1978.…”