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Comparative Study
. 2005 Jun;6(6):551-7.
doi: 10.1038/sj.embor.7400418.

Mismatch repair participates in error-free processing of DNA interstrand crosslinks in human cells

Qi Wu  1 , Laura A Christensen, Randy J Legerski, Karen M Vasquez
Affiliations
Comparative Study

Mismatch repair participates in error-free processing of DNA interstrand crosslinks in human cells

Qi Wu et al. EMBO Rep. 2005 Jun.

Abstract

DNA interstrand crosslinks (ICLs) present formidable blocks to DNA metabolic processes and must be repaired for cell survival. ICLs are induced in DNA by intercalating compounds such as the widely used therapeutic agent psoralen. In bacteria, both nucleotide excision repair (NER) and homologous recombination are required for the repair of ICLs. The processing of ICLs in mammalian cells is not clearly understood. However, it is known that processing can occur by NER, which for psoralen ICLs can be an error-generating process conducive to mutagenesis. We show here that another repair pathway, mismatch repair (MMR), is also involved in eliminating psoralen ICLs in human cells. MMR deficiency renders cells hypersensitive to psoralen ICLs without diminishing their mutagenic potential, suggesting that MMR does not contribute to error-generating repair, and that MMR may represent a relatively error-free mechanism for processing these lesions in human cells. Thus, enhancement of MMR relative to NER may reduce the mutagenesis caused by DNA ICLs in humans.

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Figures

Figure 1
Figure 1
Sensitivity of MMR-deficient and NER-deficient cells to psoralen ICLs. Survival curves are shown for cells treated with HMT and UVA at 365 nm (to allow ICL formation). (A) MSH2+ (HEC59+Chr2), MSH2− (HEC59). (B) XPA+ (GM05566), XPA− (GM05509C). Cell survival was determined by an MTT assay performed in triplicate. The bars represent the standard error of the means.
Figure 2
Figure 2
Repair efficiency of TFO-targeted psoralen ICLs in MMR-deficient and MMR-proficient human cell extracts. (A) Autoradiogram (upper panel) and ethidium-bromidestained gel (lower panel) showing DNA repair efficiencies measured by incorporation of radiolabelled nucleotides into the damaged plasmid. pSupFG1 is the damaged plasmid and pIND-lacZ is an undamaged control plasmid. (B) Quantification of repair efficiency.
Figure 3
Figure 3
Psoralen-induced mutagenesis in MMR-deficient and MMR-proficient human cell lines. MMR-proficient (HEC59+Chr2) and MMR-deficient cell lines (HEC59, LoVo) were transfected with the pSupFG1 mutation reporter plasmid and mutations in the supF reporter gene were measured 48 h after transfection. pSupFG1−UVA represents undamaged plasmid in the absence of irradiation; pSupFG1+UVA represents undamaged plasmid in the presence of UVA irradiation at 1.8 J/cm2; pAG30+UVA represents pSupFG1 plasmid treated with the psoralen-modified TFO (pAG30) and then UVA irradiation to produce psoralen ICLs in the supF mutation reporter gene; and pSCR30 represents plasmid that was incubated with the psoralen-modified control (pSCR30) oligonucleotide and UVA irradiated. The mutation frequency of the supF gene was determined as the number of mutant colonies (white colonies) to the total colonies (blue+white colonies).
Figure 4
Figure 4
Characterization of psoralen-induced mutations in human cells. Mutation spectrum of the psoralen ICL-induced mutations in the supF gene in the (A) MSH2-proficient HEC59+Chr2 cell line and (B) MSH2-deficient HEC59 cell line. Base substitutions are listed above the supFG1 sequence. Single-base deletions are indicated by a ‘–'. Multiple mutations in the same plasmid are underlined. The TFO-binding site is underlined. The targeted TA-crosslinking site is indicated by boldface type.
Figure 5
Figure 5
Model for psoralen ICL repair in human cells involving nucleotide excision repair, mismatch repair and homologous recombination.
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