Effect of DNA Methylation on the 3'→5' Exonuclease Activity of Major Human Abasic Site Endonuclease APEX1.

Apurinic/apyrimidinic (AP) endonucleases are the key enzymes in the DNA base excision repair, as they hydrolyze the phosphodiester bond in the AP site formed after removal of the damaged base. Major human AP endonuclease APEX1 also possesses the 3'-phosphodiesterase and 3'→5' exonuclease activities. The biological role of the latter has not been established yet; it is assumed that it corrects DNA synthesis errors during DNA repair. If DNA is damaged at the 3'-side of 5-methylcytosine (mC) residue, the 3'→5' exonuclease activity can change the epigenetic methylation status of the CpG dinucleotide. It remains unclear whether the 3'→5' exonuclease activity of APEX1 contributes to the active epigenetic demethylation or, on the contrary, is limited in the case of methylated CpG dinucleotides in order to preserve the epigenetic status upon repair of accidental DNA damage. Here, we report the results of the first systematic study on the efficiency of removal of 3'-terminal nucleotides from the substrates modeling DNA repair intermediates in the CpG dinucleotides. The best substrates for the 3'→5' exonuclease activity of APEX1 were oligonucleotides with the 3'-terminal bases non-complementary to the template, while the worst substrates contained mC. The presence of mC in the complementary strand significantly reduced the reaction rate even for the non-complementary 3'-ends. Therefore, the efficiency of the 3'→5' exonuclease reaction catalyzed by APEX1 is limited in the case of the methylated CpG dinucleotides, which likely reflects the need to preserve the epigenetic status during DNA repair.

Molecular dynamics approach to identification of new OGG1 cancer-associated somatic variants with impaired activity.

DNA of living cells is always exposed to damaging factors. To counteract the consequences of DNA lesions, cells have evolved several DNA repair systems, among which base excision repair is one of the most important systems. Many currently used antitumor drugs act by damaging DNA, and DNA repair often interferes with chemotherapy and radiotherapy in cancer cells. Tumors are usually extremely genetically heterogeneous, often bearing mutations in DNA repair genes. Thus, knowledge of the functionality of cancer-related variants of proteins involved in DNA damage response and repair is of great interest for personalization of cancer therapy. Although computational methods to predict the variant functionality have attracted much attention, at present, they are mostly based on sequence conservation and make little use of modern capabilities in computational analysis of 3D protein structures. We have used molecular dynamics (MD) to model the structures of 20 clinically observed variants of a DNA repair enzyme, 8-oxoguanine DNA glycosylase. In parallel, we have experimentally characterized the activity, thermostability, and DNA binding in a subset of these mutant proteins. Among the analyzed variants of 8-oxoguanine DNA glycosylase, three (I145M, G202C, and V267M) were significantly functionally impaired and were successfully predicted by MD. Alone or in combination with sequence-based methods, MD may be an important functional prediction tool for cancer-related protein variants of unknown significance.