Optimization of Deoxyinosine in vitro repair system in Human cell
Date Issued
2015
Date
2015
Author(s)
Hunag, Teng-Yung
Abstract
DNA base deamination can occur spontaneously under physiological conditions. Deamination of adenine would generate the highly mutagenic lesion, hypoxanthine(Hx) and the process is enhanced by ROS released upon exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat. Hypoxanthine in DNA can pair with cytosine which results in A:T to G:C transition mutations after DNA replication. In human cells, alkyadenine-DNA glycosylase (AAG) was thought mainly responsible for the recognition and excision of hypoxanthine. In Escherichia coli, deoxyinosine (hypoxanthine deoxyribonucleotide) is removed through an alternative excision repair pathway initiated by endonuclease V. Endo V homolog had been found in mouse and human cells, and biochemical experiment showed that mouse endo V also initiates DNA repair by endonuclease cleavage at the second phosphodieaster bond 3’ to the dI lesion. However, the correction of dI in mammalian cells appeared to be more complex and was not fully understood. To find out what machenisms may involve in dI repair in human cell, we designed dI in vitro repair system using human cell extract. In this study, we applied C-I and G-I substrates to explore the repair pathway and optimize the dI in vitro repair system in human cells. Our results showed that C-I substrate could be repaired more efficiently in HeLaS3 than in HCT116. We also tested the factor requirement for the repair in human cell extracts. Mg2+ is essential in the repair of C-I, and repair levels decreased in cell extracts without dNTPs or ATP or replaced ATP with ATP-γ-S. We concluded that ATP will be utilized and also hydrolyzed for repair process of dI substrates. We assume that DNA Polβ involved not only in BER but also in human endonuclease V-mediated excision repair, so we performed the reaction in the presence of polβ inhibitor, lithicolic acid(LCA). We found that repair levels of C-I substrate decreased about 60%. In the presence of polα、δ、 ε inhibitor aphidicolin (APH), repair levels of C-I decreased about 30% in HeLaS3 extract. HCT116 extract showed lower repair levels of dI than that of HeLaS3 extract, we believed that MMR system played some roles in dI repair system in human cell. We added purified MutLα in repair reaction and found that the deficiency of G-I repair can be partially restored by MutL-α complementation in HCT116 extract. Through these data, we concluded that there were multiple repair systems involved in dI repair, and took charge in different dI pair lesions. According to our previous study, G-I was the best substrate in HeLaS3 extract, threrfore we used G-I as substrate to optimize in vitro repair assay. We found most of substrates could be repair in 30 minutes. The opitimal concentration for ATP is 1 mM. However ATP concentration higher than 2 mM would cause G-I substrates unstable. When ATP was replaced by GTP, the repair level dramatically dropped. With Mg2+ titration test we realized too much Mg2+ would inhibit G-I repair efficiency. The opitimized dI in vitro assay will be very useful for future investigation of this important DNA repair mechanism.
Subjects
deoxyinosine repair
mismatch repair
human cell extracts
in vitro assay
DNA repair deficiency
endonuclease
Type
thesis
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