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Selective recognition of a cisplatin-DNA adduct by human mismatch repair proteins
1Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK.
Abstract:
The antitumor agent cis-diamminedichloroplatinum(II) (cisplatin) introduces cytotoxic DNA damage predominantly in the form of intrastrand crosslinks between adjacent purines. Binding assays using a series of duplex oligonucleotides containing a single 1,2 diguanyl intrastrand crosslink indicate that human cell extracts contain factors that preferentially recognise this type of damage when the complementary strand contains T opposite the 3', and C opposite the 5'guanine in the crosslink. Under the conditions of the band-shift assay used, little binding is observed if the positions of the T and C are reversed in the complementary strand. Similarly, duplexes containing CC or TT opposite the crosslink are recognised relatively poorly. The binding activity is absent from extracts of the colorectal carcinoma cell lines LoVo and DLD-1 in which the hMutSalpha mismatch recognition complex is inactivated by mutation. Extensively purified human hMutSalpha exhibits the same substrate preference and binds to the mismatched platinated DNA at least as well as to an identical unplatinated duplex containing a single G.T mismatch. It is likely, therefore, that human mismatch repair may be triggered by 1,2 diguanyl intrastrand crosslinks that have undergone replicative bypass.
Insights
Human cell extracts recognize specific DNA damage caused by the chemotherapy drug cisplatin. This recognition is linked to the hMutSalpha mismatch repair complex, suggesting a role in DNA repair after replication bypass.
Area of Science:
- Molecular Biology
- Cancer Research
- DNA Damage and Repair
Background:
- Cisplatin is a crucial chemotherapy drug that induces DNA damage, primarily intrastrand crosslinks between adjacent purines.
- Understanding the cellular mechanisms that recognize and respond to cisplatin-induced DNA damage is vital for cancer therapy.
- The hMutSalpha complex is a key player in DNA mismatch repair, but its role in recognizing drug-induced DNA adducts is less understood.
Purpose of the Study:
- To investigate the DNA binding preferences of human cell extracts towards cisplatin-induced DNA damage.
- To determine the role of the hMutSalpha complex in recognizing specific cisplatin adducts.
- To explore the potential link between DNA mismatch repair and the cellular response to cisplatin DNA damage.
Main Methods:
- Utilized band-shift assays with synthesized oligonucleotides containing specific 1,2-diguanine intrastrand crosslinks.
- Tested the binding affinity of human cell extracts and purified hMutSalpha to various platinated and unplatinated DNA duplexes.
- Compared binding activities in cell extracts from normal and mismatch repair-deficient colorectal carcinoma cell lines (LoVo, DLD-1).
Main Results:
- Human cell extracts preferentially bind to DNA with a 1,2-diguanine intrastrand crosslink when the complementary strand has T opposite the 3'-guanine and C opposite the 5'-guanine.
- Binding is significantly reduced when the positions of T and C are reversed or when CC or TT are opposite the crosslink.
- The binding activity was absent in colorectal carcinoma cell lines with inactivated hMutSalpha, and purified hMutSalpha showed similar substrate preference.
- Purified hMutSalpha binds to platinated DNA adducts as effectively as to a G.T mismatch in un-repaired DNA.
Conclusions:
- Human cell extracts possess factors that specifically recognize 1,2-diguanine intrastrand crosslinks induced by cisplatin.
- The hMutSalpha complex is likely responsible for this recognition, indicating a role in processing cisplatin DNA damage.
- DNA mismatch repair pathways, particularly involving hMutSalpha, may be activated by cisplatin adducts that escape repair and undergo replicative bypass.