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We investigate the role of the other missense mutations in DNMT3A catalytic domain found in AML using accordingly mutated murine Dnmt3a catalytic domain (Dnmt3a-CD) and 30-mer CpG-containing DNA substrates as model system. In vitro methylation assays showed the 3-5-fold reduced activity for R181L (R771L), S124C (S714C) and P187R (P777R) mutants. The most pronounced reduction of the activty was observed for F152V (F752V), R45W (R635W) and R146H (R736H). Further, the effect of these mutations on individual steps of the methylation reaction was studied. R181L (R771L), S124C (S714C) and P187R (P777R) preserve the ability to bind DNA as it was shown by similar dissociation constants for the Dnmt3a-CD/DNA complexes. In the case of R45W (R635W) and R146H (R736H) a complete loss of DNA binding properties was observed. Finally, the ability of the DNMT3A partner protein DNMT3L to restore the methylation activities of S124C (S714C) and R181L (R771L) was revealed. Hence, mutation in DNMT3A leads to diverse levels of activity and interaction with Dnmt3a partners. Strikingly, all the mutations except S124C (S714C) are not located in the DNMT3A catalytic loop. The contribution of the studied specific residues to molecular mechanism of DNMT3a-mediated DNA methylation was suggested. The role of aberrant DNMT3a activity in AML was discussed on the basis of our knowledge of how these mutations affect methylation function. Collectively, these data together with previously studied R790 and R792 DNMT3a mutants suggest functional impairment of DNMT3a during pathogenesis.
№ | Имя | Описание | Имя файла | Размер | Добавлен |
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1. | Biokataliz_2019.pdf | Biokataliz_2019.pdf | 357,7 КБ | 28 декабря 2019 [KirsanovaOV] |