The Escherichia coli mismatch repair protein MutL recruits the Vsr and MutH endonucleases in response to DNA damage.

The activities of the Vsr and MutH endonucleases of Escherichia coli are stimulated by MutL. The interaction of MutL with each enzyme is enhanced in vivo by 2-aminopurine treatment and by inactivation of the mutY gene. We hypothesize that MutL recruits the endonucleases to sites of DNA damage.

Interaction between the mismatch repair and nucleotide excision repair pathways in the prevention of 5-azacytidine-induced CG-to-GC mutations in Escherichia coli.

5-Azacytidine induces CG-to-GC transversion mutations in Escherichia coli. The results presented in this paper provide evidence that repair of the drug-induced lesions that produce these mutations involves components of both the mismatch repair and nucleotide excision repair systems. Strains deficient in mutL, mutS, uvrA, uvrB or uvrC all showed an increase in mutation in response to 5-azacytidine. Using a bacterial two-hybrid assay, we showed that UvrB interacts with MutL and MutS in a drug-dependent manner, while UvrC interacts with MutL independent of drug. We suggest that 5-azacytidine-induced mismatches recruit MutS and MutL, but are poorly processed by mismatch repair. Instead, the stalled MutS-MutL complex recruits the Uvr proteins to complete repair.

Reduced host cell reactivation of oxidative DNA damage in human cells deficient in the mismatch repair gene hMSH2.

Germ line mutations in the mismatch repair (MMR) genes hMSH2 and hMLH1 account for approximately 98% of hereditary non-polyposis colorectal cancers. In addition, there is increasing evidence for an involvement of MMR gene expression in the response of cells to UV-induced skin cancer. The link between MMR and skin cancer suggests an involvement of MMR gene expression in the response of skin cells to UV-induced DNA damage. In this report, we have used two reporter gene assays to examine the role of hMSH2 and hMLH1 in the repair of oxidative DNA damage induced by UVA light and DNA damage caused by methylene blue plus visible light (MB+VL). UVA and MB+VL produce 8-hydroxyguanines in DNA that are repaired by base excision repair (BER). AdHCMVlacZ is a replication-deficient recombinant adenovirus that expresses the beta-galactosidase (beta-gal) reporter gene under the control of the human cytomegalovirus (CMV) immediate-early promoter. We show a reduced host cell reactivation for beta-gal expression of UVA-treated and MB+VL-treated AdHCMVlacZ in hMSH2-deficient LoVo human colon adenocarcinoma cells compared to their hMSH2-proficient counterpart SW480 cells, but not in hMLH1-deficient HCT116 human colon adenocarcinoma cells compared to hMLH1-proficient HCT116-chr3 cells. We have also reported previously that enhanced expression of the undamaged AdHCMVlacZ reporter gene is induced by the pre-treatment of cells with lower levels of the DNA-damaging agent and to higher expression levels in transcription-coupled repair (TCR)-deficient compared to TCR-proficient cells. Here we show that pre-treatment of cells with UVA or MB+VL enhanced expression of the undamaged reporter gene to a higher level in LoVo compared to SW480 cells but there was little or no difference in HCT116 compared to HCT116-chr3 cells. These results suggest a substantial involvement of hMSH2 but little or no involvement of hMLH1 in the repair of UVA- and MB+VL-induced oxidative DNA damage by BER.

Enhanced host cell reactivation of a UV-damaged reporter gene in pre-UV-treated cells is delayed in Cockayne syndrome cells.

We have used a non-replicating recombinant adenovirus, Ad5HCMVlacZ, which expresses the beta-galactosidase (beta-gal) reporter gene, to examine the time course of UV-inducible repair of UV-damaged DNA in human fibroblasts. Host cell reactivation (HCR) of beta-gal activity for UV-irradiated Ad5HCMVlacZ was examined in non-irradiated and UV-irradiated nucleotide excision repair (NER) proficient normal human fibroblasts, xeroderma pigmentosum (XP) group C fibroblasts which are defective in the global genomic repair (GGR) pathway of NER and Cockayne syndrome (CS) fibroblasts which are defective in the transcription coupled repair (TCR) pathway of NER. HCR was deficient in untreated XP-C and CS cells indicating that both TCR and GGR are involved in removal of photolesions from the transcribed strand of the reporter gene in unirradiated human cells as reported previously. Prior UV-irradiation of cells with low UV fluences resulted in a transient enhancement of HCR in normal and XP-C fibroblasts that reached a maximum when cells were infected at 25-35 h after UV. In contrast, UV-enhanced HCR was delayed in CS-B cells, reaching levels similar to that in normal cells only when cells were infected between 40 and 60 h after UV exposure. These results are consistent with a UV-induced up-regulation of GGR through a TCR dependent pathway in CS cells.

Detection of an involvement of the human mismatch repair genes hMLH1 and hMSH2 in nucleotide excision repair is dependent on UVC fluence to cells.

There is conflicting evidence for the role of the mismatch repair (MMR) genes hMLH1 and hMSH2 in the transcription-coupled repair (TCR) pathway of nucleotide excision repair. In the present work, we have examined the role of these MMR genes in nucleotide excision repair using two reporter gene assays. AdHCMVlacZ is a replication-deficient recombinant adenovirus that expresses the beta-galactosidase reporter gene under the control of the human cytomegalovirus immediate early promoter. We have reported previously a reduced host cell reactivation (HCR) for beta-galactosidase expression of UVC-irradiated AdHCMVlacZ in TCR-deficient Cockayne syndrome (CS) fibroblasts compared with normal fibroblasts, indicating that HCR depends, at least in part, on TCR. In addition, we have reported that UVC-enhanced expression of the undamaged reporter gene is induced at lower UVC fluences to cells and at higher levels after low UVC fluences in TCR-deficient compared with normal human fibroblasts, suggesting that persistent damage in active genes triggers increased activity from the human cytomegalovirus-driven reporter construct. We have examined HCR and UV-enhanced expression of the reporter gene in hMLH1-deficient HCT116 human colon adenocarcinoma cells and HCT116-chr3 cells (the MMR-proficient counterpart of HCT116) as well as hMSH2-deficient LoVo human colon adenocarcinoma cells and their hMSH2-proficient counterpart SW480 cells. We show a greater UV-enhanced expression of the undamaged reporter gene after low UVC exposure in HCT116 compared with HCT116-chr3 cells and in LoVo compared with SW480 cells. We show also a reduced HCR in HCT116 compared with HCT116-chr3 cells and in LoVo compared with SW480 cells. However, the reduction in HCR was less or absent when cells were pretreated with UVC. These results suggest that detection of an involvement of hMLH1 and hMSH2 in TCR is dependent on UVC (254 nm) fluence to cells.

Mechanism of 2-aminopurine-stimulated mutagenesis in Escherichia coli.

2-Aminopurine (2AP), a base analog, causes both transition and frameshift mutations in Escherichia coli. The analog is thought to cause mutations by two mechanisms: directly, by mispairing with cytosine, and indirectly, by saturation of mismatch repair (MMR). The goal of this work was to measure the relative contribution of these two mechanisms to the occurrence of transition mutations. Our data suggest that, in contrast to 2-aminopurine-stimulated frameshift mutations, the majority of transition mutations are a direct effect of base mispairing.