The enzymatic basis of processivity in λ exonuclease
The enzymatic basis of processivity in λ exonuclease
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DOI：
10.1093/nar/gkg266
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发表时间：
2003-03-15
影响因子：
14.9
通讯作者：
Myers, RS
中科院分区：
文献类型：
--
作者：
Subramanian, K;Rutvisuttinunt, W;Myers, RS
λ核酸外切酶是降解双链（ds）DNA的高度进行性5 '->3'核酸外切酶。由λ核酸外切酶产生的单链DNA被同源配对蛋白质利用以进行同源重组。对λ生物学、λ核酸外切酶酶学的广泛研究以及λ核酸外切酶的X射线晶体学结构的可用性使其成为剖析持续合成能力机制的合适模型。λ核酸外切酶是环状同源三聚体分子，并且这种四级结构是参与核酸代谢中的进行性反应的蛋白质中反复出现的主题。我们已经鉴定了λ核酸外切酶中参与识别断裂dsDNA末端的5 '-磷酸的残基。在以前的研究中已经建立了λ外切核酸酶对5' dsDNA末端磷酸部分的偏好;我们的结果表明，在不存在5'-磷酸的情况下的低活性是由于惰性酶-底物复合物的形成。通过检查在5 '-磷酸识别中受损的λ核酸外切酶突变体，已经阐明了催化效率在调节λ核酸外切酶的持续合成能力中的意义。我们提出了一个模型，在该模型中，λ核酸外切酶的持续合成能力表示为诱导正向易位或促进反向易位和解离的途径之间的竞争的净结果。
lambda Exonuclease is a highly processive 5'-->3' exonuclease that degrades double-stranded (ds)DNA. The single-stranded DNA produced by lambda exonuclease is utilized by homologous pairing proteins to carry out homologous recombination. The extensive studies of lambda biology, lambda exonuclease enzymology and the availability of the X-ray crystallographic structure of lambda exonuclease make it a suitable model to dissect the mechanisms of processivity. lambda Exonuclease is a toroidal homotrimeric molecule and this quaternary structure is a recurring theme in proteins engaged in processive reactions in nucleic acid metabolism. We have identified residues in lambda exonuclease involved in recognizing the 5'-phosphate at the ends of broken dsDNA. The preference of lambda exonuclease for a phosphate moiety at 5' dsDNA ends has been established in previous studies; our results indicate that the low activity in the absence of the 5'-phosphate is due to the formation of inert enzyme-substrate complexes. By examining a lambda exonuclease mutant impaired in 5'-phosphate recognition, the significance of catalytic efficiency in modulating the processivity of lambda exonuclease has been elucidated. We propose a model in which processivity of lambda exonuclease is expressed as the net result of competition between pathways that either induce forward translocation or promote reverse translocation and dissociation.