Elevated Levels of the Escherichia coli nrdAB -Encoded Ribonucleotide Reductase Counteract the Toxicity Caused by an Increased Abundance of the β Clamp
Elevated Levels of the Escherichia coli nrdAB -Encoded Ribonucleotide Reductase Counteract the Toxicity Caused by an Increased Abundance of the β Clamp
复制标题
大肠杆菌 nrdAB 编码核糖核苷酸还原酶水平升高可抵消 β 钳丰度增加引起的毒性
DOI：
10.1128/jb.00304-21
复制
发表时间：
2021
影响因子：
3.2
通讯作者：
Sutton, Mark D.
中科院分区：
文献类型：
--
作者：
Babu, Vignesh M.;Homiski, Caleb;Scotland, Michelle K.;Chodavarapu, Sundari;Kaguni, Jon M.;Sutton, Mark D.
大肠杆菌N编码的β钳蛋白的表达水平比染色体表达水平高≥10倍，通过干扰DNA复制来阻碍生长。突变型钳（β E202 K在残基202处具有谷氨酸至赖氨酸的取代）与DNA聚合酶III（Pol III）的结合亲和力高于野生型钳，表明其未能阻碍生长与其将Pol III与复制叉隔离的能力无关。我们的研究结果表明，由于DnaA-ATP水平不足，dnaNE 202 K菌株启动DNA复制不足，并以改变的水平表达了几个DnaA调节的基因，包括编码1a类核糖核苷酸还原酶（RNR）的nrdAB。nrdAB的高表达依赖于功能障碍。由于β-clamp-Hda复合物通过刺激其内在ATP酶活性来调节DnaA的活性，这一发现表明，与野生型菌株相比，dnaNE 202 K等位基因支持体内Hda活性水平升高。相比之下，使用用纯化组分重构的体外测定，β E202 K和野生型钳蛋白支持相当水平的Hda活性。然而，thenrdAB编码的RNR的共过表达缓解了由β钳水平升高引起的生长缺陷。这些结果支持了一个模型，在该模型中，DNA前体细胞水平的增加缓解了β钳夹水平升高阻碍生长的能力，并表明源自dnaNE 202 K突变的多种效应有助于dnrdAB水平升高，或者Hda在调节DnaA-202中起非催化作用。重要的是，与ATP结合的DnaA在DNA复制的起始中起作用，并调节DNA复制的表达。其产物在DNA代谢中起作用的几个基因。与DnaA结合的ATP的状态部分地由β clamp-Hda复合物调节。dnaNE 202 K等位基因的鉴定依据是当其表达水平高于染色体表达水平10倍以上时不能阻碍生长。虽然dnaNE 202 K菌株表现出几种与Hda活性升高一致的表型，但野生型和β E202 K夹蛋白在体外支持相同水平的Hda活性。这些结果表明β E202 K-Hda在DnaA-ATP的调节中起非催化作用。这一点，以及替代模式，进行了讨论。
Expression of the Escherichia colidnaN-encoded β clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. A mutant clamp (βE202Kbearing a glutamic acid-to-lysine substitution at residue 202) binds to DNA polymerase III (Pol III) with higher affinity than the wild-type clamp, suggesting that its failure to impede growth is independent of its ability to sequester Pol III away from the replication fork. Our results demonstrate that thednaNE202Kstrain underinitiates DNA replication due to insufficient levels of DnaA-ATP and expresses several DnaA-regulated genes at altered levels, includingnrdAB, that encode the class 1a ribonucleotide reductase (RNR). Elevated expression ofnrdABwas dependent onhdafunction. As the β clamp-Hda complex regulates the activity of DnaA by stimulating its intrinsic ATPase activity, this finding suggests that thednaNE202Kallele supports an elevated level of Hda activityin vivocompared with the wild-type strain. In contrast, using anin vitroassay reconstituted with purified components the βE202Kand wild-type clamp proteins supported comparable levels of Hda activity. Nevertheless, co-overexpression of thenrdAB-encoded RNR relieved the growth defect caused by elevated levels of the β clamp. These results support a model in which increased cellular levels of DNA precursors relieve the ability of elevated β clamp levels to impede growth and suggest either that multiple effects stemming from thednaNE202Kmutation contribute to elevatednrdABlevels or that Hda plays a noncatalytic role in regulating DnaA-ATP by sequestering it to reduce its availability.IMPORTANCEDnaA bound to ATP acts in initiation of DNA replication and regulates the expression of several genes whose products act in DNA metabolism. The state of the ATP bound to DnaA is regulated in part by the β clamp-Hda complex. ThednaNE202Kallele was identified by virtue of its inability to impede growth when expressed ≥10-fold higher than chromosomally expressed levels. While thednaNE202Kstrain exhibits several phenotypes consistent with heightened Hda activity, the wild-type and βE202Kclamp proteins support equivalent levels of Hda activityin vitro. Taken together, these results suggest that βE202K-Hda plays a noncatalytic role in regulating DnaA-ATP. This, as well as alternative models, is discussed.