New England Biolabs, Inc., Ipswich, Massachusetts, United States of America.
PLoS One. 2010 Jul 26;5(7):e11787. doi: 10.1371/journal.pone.0011787.
The Type IIS restriction endonuclease BtsI recognizes and digests at GCAGTG(2/0). It comprises two subunits: BtsIA and BtsIB. The BtsIB subunit contains the recognition domain, one catalytic domain for bottom strand nicking and part of the catalytic domain for the top strand nicking. BtsIA has the rest of the catalytic domain that is responsible for the DNA top strand nicking. BtsIA alone has no activity unless it mixes with BtsIB to reconstitute the BtsI activity. During characterization of the enzyme, we identified a BtsIB mutant R119A found to have a different digestion pattern from the wild type BtsI. After characterization, we found that BtsIB(R119A) is a novel restriction enzyme with a previously unreported recognition sequence CAGTG(2/0), which is named as BtsI-1. Compared with wild type BtsI, BtsI-1 showed different relative activities in NEB restriction enzyme reaction buffers NEB1, NEB2, NEB3 and NEB4 and less star activity. Similar to the wild type BtsIB subunit, the BtsI-1 B subunit alone can act as a bottom nicking enzyme recognizing CAGTG(-/0). This is the first successful case of a specificity change among this restriction endonuclease type.
II 型限制内切酶 BtsI 识别并消化 GCAGTG(2/0)。它由两个亚基组成：BtsIA 和 BtsIB。BtsIB 亚基包含识别结构域、一个用于底部链切口的催化结构域和部分用于顶部链切口的催化结构域。BtsIA 具有负责 DNA 顶部链切口的剩余催化结构域。除非 BtsIA 与 BtsIB 混合以重新构成 BtsI 活性，否则 BtsIA 本身没有活性。在对该酶进行表征的过程中，我们鉴定出一个 BtsIB 突变体 R119A，其消化模式与野生型 BtsI 不同。经过表征，我们发现 BtsIB(R119A)是一种新型限制酶，具有以前未报道的识别序列 CAGTG(2/0)，命名为 BtsI-1。与野生型 BtsI 相比，BtsI-1 在 NEB 限制酶反应缓冲液 NEB1、NEB2、NEB3 和 NEB4 中表现出不同的相对活性和较少的星活性。与野生型 BtsIB 亚基类似，BtsI-1 的 B 亚基单独可以作为识别 CAGTG(-/0)的底部切口酶。这是该限制内切酶类型中首次成功改变特异性的案例。
