Janscak P, Sandmeier U, Bickle T A
Department of Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056, Switzerland.
Nucleic Acids Res. 1999 Jul 1;27(13):2638-43. doi: 10.1093/nar/27.13.2638.
Type I restriction enzymes bind to specific DNA sequences but subsequently translocate non-specific DNA past the complex in a reaction coupled to ATP hydrolysis and cleave DNA at any barrier that can halt the translocation process. The restriction subunit of these enzymes, HsdR, contains a cluster of seven amino acid sequence motifs typical of helicase superfamily II, that are believed to be relevant to the ATP-dependent DNA translocation. Alignment of all available HsdR sequences reveals an additional conserved region at the protein N-terminus with a consensus sequence reminiscent of the P-D.(D/E)-X-K catalytic motif of many type II restriction enzymes. To investigate the role of these conserved residues, we have produced mutants of the type IB restriction enzyme Eco AI. We have found that single alanine substitutions at Asp-61, Glu-76 and Lys-78 residues of the HsdR subunit abolished the enzyme's restriction activity but had no effect on its ATPase and DNA translocation activities, suggesting that these residues are part of the active site for DNA cleavage.
I型限制酶与特定的DNA序列结合，但随后在与ATP水解偶联的反应中使非特异性DNA绕过复合物移位，并在任何能阻止移位过程的屏障处切割DNA。这些酶的限制亚基HsdR包含一组七个典型的解旋酶超家族II氨基酸序列基序，据信与ATP依赖性DNA移位有关。所有可用的HsdR序列比对显示，在蛋白质N端有一个额外的保守区域，其共有序列让人联想到许多II型限制酶的P-D.(D/E)-X-K催化基序。为了研究这些保守残基的作用，我们构建了I型B限制酶Eco AI的突变体。我们发现，HsdR亚基的Asp-61、Glu-76和Lys-78残基处的单个丙氨酸取代消除了该酶的限制活性，但对其ATP酶和DNA移位活性没有影响，这表明这些残基是DNA切割活性位点的一部分。
