Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA.
Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA.
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DOI：
10.1093/nar/gks914
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发表时间：
2012-12
影响因子：
14.9
通讯作者：
Klimasauskas S
中科院分区：
文献类型：
--
作者：
Lukinavicius G;Lapinaite A;Urbanaviciute G;Gerasimaite R;Klimasauskas S
甲基转移酶催化普遍存在的辅因子S-腺苷-L-蛋氨酸(ADO-Met)的甲基转移到DNA的特定靶点上，在从细菌到人类的生物体中发挥着重要的作用。尽管与合成类似物的反应效率仍然很低，但人们已经在化学上合成了带有延长的丙醇酸侧链的ADOMet类似物，用于甲基转移酶直接将激活基团转移到DNA上。我们对模式DNA胞嘧啶-5甲基转移酶(C5-MTase)M.HhaI中的辅因子口袋进行了立体工程，系统地替换了位于两个保守序列基序和可变区的三个非必要位置，并具有较小的残基。我们发现，二重和三重取代导致了烷基转移活性的显著提高，表现为辅因子结合亲和力略有增加，烷基转移速率增加较大。伴随这些效应的是产物DNA-M.HhaI-ADOHcy复合体的稳定性和甲基化速率的降低，从而允许在ADOMet存在的情况下进行竞争性mTAG标记。对M.HpaII和M2.Eco31I中两个保守残基的类似替换也导致了烷基转移活性的提高，证明了同源性指导的工程对C5-MTase家族的普遍适用性，并扩大了用于体外和体外标记DNA的序列特异性工具的库。
DNA methyltransferases catalyse the transfer of a methyl group from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) onto specific target sites on DNA and play important roles in organisms from bacteria to humans. AdoMet analogs with extended propargylic side chains have been chemically produced for methyltransferase-directed transfer of activated groups (mTAG) onto DNA, although the efficiency of reactions with synthetic analogs remained low. We performed steric engineering of the cofactor pocket in a model DNA cytosine-5 methyltransferase (C5-MTase), M.HhaI, by systematic replacement of three non-essential positions, located in two conserved sequence motifs and in a variable region, with smaller residues. We found that double and triple replacements lead to a substantial improvement of the transalkylation activity, which manifests itself in a mild increase of cofactor binding affinity and a larger increase of the rate of alkyl transfer. These effects are accompanied with reduction of both the stability of the product DNA–M.HhaI–AdoHcy complex and the rate of methylation, permitting competitive mTAG labeling in the presence of AdoMet. Analogous replacements of two conserved residues in M.HpaII and M2.Eco31I also resulted in improved transalkylation activity attesting a general applicability of the homology-guided engineering to the C5-MTase family and expanding the repertoire of sequence-specific tools for covalent in vitro and ex vivo labeling of DNA.
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影响因子：
14.9
作者：
Sukackaite R;Grazulis S;Tamulaitis G;Siksnys V
通讯作者：
Siksnys V
影响因子：
14.9
作者：
Gerasimaite R;Vilkaitis G;Klimasauskas S
通讯作者：
Klimasauskas S
影响因子：
14.9
作者：
Bujnicki, JM;Feder, M;Redman, KL
通讯作者：
Redman, KL
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作者：
Neely, Robert K.;Dedecker, Peter;Hofkens, Johan
通讯作者：
Hofkens, Johan
影响因子：
4.7
作者：
Braun, Gary;Diechrierow, Michael;Reich, Norbert O.
通讯作者：
Reich, Norbert O.