Williams S A, Halford S E
Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
Nucleic Acids Res. 2001 Apr 1;29(7):1476-83. doi: 10.1093/nar/29.7.1476.
The SfiI endonuclease cleaves DNA at the sequence GGCCNNNN NGGCC, where N is any base and downward arrow is the point of cleavage. Proteins that recognise discontinuous sequences in DNA can be affected by the unspecified sequence between the specified base pairs of the target site. To examine whether this applies to SFII, a series of DNA duplexes were made with identical sequences apart from discrete variations in the 5 bp spacer. The rates at which SFII cleaved each duplex were measured under steady-state conditions: the steady-state rates were determined by the DNA cleavage step in the reaction pathway. SFII cleaved some of these substrates at faster rates than other substrates. For example, the change in spacer sequence from AACAA to AAACA caused a 70-fold increase in reaction rate. In general, the extrapolated values for k(cat) and K(m) were both higher on substrates with inflexible spacers than those with flexible structures. The dinucleotide at the site of cleavage was largely immaterial. SFII activity is thus highly dependent on conformational variations in the spacer DNA.
SfiI核酸内切酶在序列GGCCNNNN NGGCC处切割DNA，其中N为任意碱基，向下的箭头表示切割点。识别DNA中不连续序列的蛋白质可能会受到靶位点特定碱基对之间未指定序列的影响。为了研究这是否适用于SFII，制备了一系列DNA双链体，它们除了5个碱基对的间隔区存在离散变化外，序列相同。在稳态条件下测量了SFII切割每个双链体的速率：稳态速率由反应途径中的DNA切割步骤决定。SFII切割其中一些底物的速率比其他底物快。例如，间隔区序列从AACAA变为AAACA导致反应速率提高了70倍。一般来说，与具有柔性结构的底物相比，具有刚性间隔区的底物的k(cat)和K(m)的外推值都更高。切割位点处的二核苷酸在很大程度上无关紧要。因此，SFII活性高度依赖于间隔区DNA的构象变化。
