Wang Jianlei, Julin Douglas A
Department of Chemistry & Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
J Biol Chem. 2004 Dec 10;279(50):52024-32. doi: 10.1074/jbc.M408645200. Epub 2004 Oct 4.
The bacterium Deinococcus radiodurans is extremely resistant to high levels of DNA-damaging agents, including gamma rays and ultraviolet light that can lead to double-stranded DNA breaks. Surprisingly, the organism does not appear to have a RecBCD enzyme, an enzyme that is critical for double-strand break repair in many other bacteria. The D. radiodurans genome does encode a protein whose closest characterized homologues are RecD subunits of RecBCD enzymes in other bacteria. We have purified this novel D. radiodurans RecD protein and characterized its biochemical activities. The D. radiodurans RecD protein is a DNA helicase that unwinds short (20 base pairs) DNA duplexes with either a 5'-single-stranded tail or a forked end, but not blunt-ended or 3'-tailed duplexes. Duplexes with 10-12 nucleotide (nt) 5'-tails are good unwinding substrates and are bound tightly, while DNA with shorter tails (4-8 nt) are poor unwinding substrates and are bound much less tightly. The RecD protein is much less efficient at unwinding slightly longer substrates (52 or 76 base pairs, with 12 nt 5'-tails). Unwinding of the longer substrates is stimulated somewhat (4-5-fold) by the single-stranded DNA-binding protein from D. radiodurans. These results show that the D. radiodurans RecD protein is a DNA helicase with 5'-3' polarity and low processivity.
耐辐射球菌对高水平的DNA损伤剂具有极强的抗性，这些损伤剂包括可导致双链DNA断裂的伽马射线和紫外线。令人惊讶的是，该生物体似乎没有RecBCD酶，而这种酶在许多其他细菌的双链断裂修复中至关重要。耐辐射球菌的基因组确实编码一种蛋白质，其最具特征的同源物是其他细菌中RecBCD酶的RecD亚基。我们已经纯化了这种新型的耐辐射球菌RecD蛋白，并对其生化活性进行了表征。耐辐射球菌RecD蛋白是一种DNA解旋酶，可解开具有5'-单链尾巴或叉状末端的短（20个碱基对）DNA双链体，但不能解开平头或3'-尾双链体。具有10 - 12个核苷酸（nt）5'-尾巴的双链体是良好的解旋底物，并且紧密结合，而具有较短尾巴（4 - 8 nt）的DNA是较差的解旋底物，结合紧密程度低得多。RecD蛋白解旋稍长一些的底物（52或76个碱基对，带有12 nt 5'-尾巴）的效率要低得多。耐辐射球菌的单链DNA结合蛋白对较长底物的解旋有一定程度的刺激（4 - 5倍）。这些结果表明，耐辐射球菌RecD蛋白是一种具有5'-3'极性和低持续性的DNA解旋酶。
