Functional studies of the small subunit of EcoHK31I DNA methyltransferase
Functional studies of the small subunit of EcoHK31I DNA methyltransferase
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DOI：
10.1515/bc.2006.066
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发表时间：
2006-05-01
影响因子：
3.7
通讯作者：
Shaw, Pang-Chui
中科院分区：
文献类型：
--
作者：
Fung, Wai-To;Sze, Kong-Hung;Shaw, Pang-Chui
EcoHK311 DNA 甲基转移酶可识别序列 5'-YGGCCR-3'，并将甲基添加到内部胞嘧啶的第五个位置，以保护 DNA 免受其同源核酸内切酶的切割。 M.EcoHK311由多肽α和β组成。 Polypepticle beta仅含有C5-MTase家族的保守IX基序，并提供了一个独特的例子来表明该基序单独可能被移位到另一个多肽上。通过电迁移率测定，我们发现α/β复合物识别特定的寡核苷酸底物。多肽α与DNA形成聚集体，而单独的多肽p不结合DNA。因此，多肽β有助于多肽α与DNA底物的正确结合。多肽α和N端缺失41个氨基酸的多肽β变体的复合物显示甲基化活性降低了16倍。进一步缺失导致甲基转移酶失活。 α/β复合物的解离平衡常数(K-d)为56.4nM，而α/Delta N46-多肽β复合物的K-d值增加了约95倍，这是由于解离速率常数(k(d))的急剧下降和缔合速率常数(k(a))的增加造成的。这表明多肽β的N端区域参与亚基相互作用，而C端区域参与DNA结合。
EcoHK311 DNA methyltransferase recognizes the sequence 5'-YGGCCR-3' and adds a methyl group to the fifth position of the internal cytosine to protect the DNA from cleavage by its cognate endonuclease. M.EcoHK311 is composed of polypeptides alpha and beta. Polypepticle beta only contains the conserved IX motif of the C5-MTase family, and provides a unique example to show that this motif alone may be dislocated to another polypeptide. By electromobility shift assay, we found that the alpha/beta complex recognizes specific oligonucleotide substrates. Polypeptide alpha formed aggregates with DNA, while polypeptide p alone did not bind DNA. Therefore, polypeptide beta assists in the proper binding of polypeptide alpha to DNA substrate. The complex of polypeptide alpha and a polypeptide beta variant with an N-terminal deletion of 41 amino acids showed a 16-fold reduction in methylation activity. Further deletion resulted in an inactive methyltransferase. The dissociation equilibrium constant (K-d) of the alpha/beta complex was 56.4 nM, while the K-d value for the alpha/Delta N46-polypeptide beta complex was increased approximately 95-fold, caused by a drastic decrease in dissociate rate constant (k(d)) and an increase in the association rate constant (k(a)). This indicates that the N-terminal region of polypeptide beta takes part in subunit interaction, while the C-terminal region is involved in DNA binding.