School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
Biochimie. 2018 Mar;146:1-13. doi: 10.1016/j.biochi.2017.11.002. Epub 2017 Nov 10.
The enzyme T4 endonuclease VII is a resolvase that acts on branched DNA intermediates during genetic recombination, by cleaving DNA with staggered cuts approximately 3-6 bp apart. In this paper, we investigated the sequence preference of this cleavage reaction utilising two different DNA sequences. For the first time, the DNA sequence preference of T4 endonuclease VII cleavage sites has been examined without the presence of a known DNA substrate to mask any inherent nucleotide preference. The use of the ABI3730 platform enables the cleavage site to be determined at nucleotide resolution. We found that T4 endonuclease VII cleaves DNA with a sequence preference. We calculated the frequency of nucleotides surrounding the cleavage sites and found that following nucleotides had the highest incidence: AWTANSTC, where N indicates the cleavage site between positions 0 and 1, N is any base, W is A or T, and S is G or C. An A at position -1 and T at position +2 were the most predominant nucleotides at the cleavage site. Using a Sequence Logo method, the sequence TATTAN*CT was derived at the cleavage site. Note that A and T nucleotides were highly preferred 5' to the cleavage sites in both methods of analysis. It was proposed that the enzyme recognises the narrower minor groove of these consecutive AT base pairs and cleaves DNA 3' to this feature.
T4 内切核酸酶 VII 是一种在基因重组过程中作用于分支 DNA 中间体的解旋酶，通过在大约 3-6 bp 处交错切割 DNA 进行切割。在本文中，我们利用两种不同的 DNA 序列研究了这种切割反应的序列偏好。首次在没有已知 DNA 底物掩盖任何固有核苷酸偏好的情况下检查了 T4 内切核酸酶 VII 切割位点的 DNA 序列偏好。ABI3730 平台的使用能够以核苷酸分辨率确定切割位点。我们发现 T4 内切核酸酶 VII 切割 DNA 具有序列偏好。我们计算了切割位点周围核苷酸的频率，发现以下核苷酸的发生率最高：AWTANSTC，其中 N表示位置 0 和 1 之间的切割位点，N 是任何碱基，W 是 A 或 T，S 是 G 或 C。在切割位点处，A 位于-1 位，T 位于+2 位，是最主要的核苷酸。使用序列徽标方法，在切割位点处得到了 TATTAN*CT 序列。请注意，在两种分析方法中，A 和 T 核苷酸在切割位点的 5'端都高度优先。有人提出，该酶识别这些连续 AT 碱基对较窄的小沟，并在该特征的 3'端切割 DNA。
