Sliding clamp-DNA interactions are required for viability and contribute to DNA polymerase management in Escherichia coli.
Sliding clamp-DNA interactions are required for viability and contribute to DNA polymerase management in Escherichia coli.
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DOI：
10.1016/j.jmb.2009.01.050
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发表时间：
2009-03-20
影响因子：
5.6
通讯作者：
Sutton, Mark D.
中科院分区：
文献类型：
--
作者：
Heltzel, Justin M. H.;Ponticelli, Sarah K. Scouten;Sanders, Laurie H.;Duzen, Jill M.;Cody, Vivian;Pace, James;Snell, Edward H.;Sutton, Mark D.
滑动钳蛋白在拓扑学上包围DNA，并在协调各种DNA复制、修复和损伤耐受蛋白的作用中起重要作用。E.大肠杆菌β钳与它拓扑包围的DNA物理相互作用。我们利用携带G66 E和G174 A取代（β159）的突变β夹蛋白，影响单链（ss）DNA结合区，或多聚丙氨酸取代残基148-HQDVR-152（β148-152），影响双链（ds）DNA结合区，以确定夹-DNA相互作用的生物学相关性。作为这项工作的一部分，我们解决了β148-152的X射线晶体结构，这证实了聚丙氨酸取代未能显著改变钳的三级结构。基于功能测定，β159和β148-152在体外在线性引发的DNA上的负载和保留均受损。在β148-152的情况下，这种缺陷不是由于与DnaX夹加载器的相互作用改变，而是β148-152-DNA相互作用受损的结果。一旦加载，β148-152在体外对Pol III复制是熟练的。相比之下，β148-152在Pol II和Pol IV复制中严重受损，并且在与这些Pol的直接物理相互作用中类似地受损。尽管β148-152能够支持Pol III的体外复制，但它不能支持E.杆菌然而，从质粒表达的生理水平的β148-152有效地补充了表达β159（dnaN 159）的菌株的温度敏感性生长表型，条件是Pol II和Pol IV被灭活。虽然该菌株的Pol V依赖性诱变受损，但Pol II和Pol IV的失活恢复了Pol V突变子表型。总之，这些结果支持了一个模型，其中竞争性clamp-DNA，clamp-partner和partner-DNA相互作用的复杂组合用于管理不同E. coliPos体内表达。
Sliding clamp proteins topologically encircle DNA and play vital roles in coordinating the actions of various DNA replication, repair, and damage tolerance proteins. At least three distinct surfaces of the E. coli β clamp interact physically with the DNA that it topologically encircles. We utilized mutant β clamp proteins bearing G66E and G174A substitutions (β159), affecting the single strand (ss) DNA-binding region, or poly-Ala substitutions in place of residues 148-HQDVR-152 (β148–152), affecting the double strand (ds) DNA binding region, in order to determine the biological relevance of clamp-DNA interactions. As part of this work, we solved the x-ray crystal structure of β148–152, which verified that the poly-Ala substitutions failed to significantly alter the tertiary structure of the clamp. Based on functional assays, both β159 and β148–152 were impaired for loading and retention on a linear primed DNA in vitro. In the case of β148–152, this defect was not due to altered interactions with the DnaX clamp loader, but rather was the result of impaired β148–152-DNA interactions. Once loaded, β148–152 was proficient for Pol III replication in vitro. In contrast, β148–152 was severely impaired for Pol II and Pol IV replication, and was similarly impaired for direct physical interactions with these Pols. Despite its ability to support Pol III replication in vitro, β148–152 was unable to support viability of E. coli. Nevertheless, physiological levels of β148–152 expressed from a plasmid efficiently complemented the temperature sensitive growth phenotype of a strain expressing β159 (dnaN159), provided that Pol II and Pol IV were inactivated. Although this strain was impaired for Pol V-dependent mutagenesis, inactivation of Pol II and Pol IV restored the Pol V mutator phenotype. Taken together, these results support a model in which a sophisticated combination of competitive clamp-DNA, clamp-partner, and partner-DNA interactions serve to manage the actions of the different E. coli Pols in vivo.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
64.5
作者：
Jeruzalmi, D;Yurieva, O;Kuriyan, J
通讯作者：
Kuriyan, J
影响因子：
64.5
作者：
KONG, XP;ONRUST, R;KURIYAN, J
通讯作者：
KURIYAN, J
影响因子：
5.6
作者：
Burnouf, DY;Olieric, V;Dumas, P
通讯作者：
Dumas, P
DOI：
10.1073/pnas.0506430102
发表时间：
2005-09-27
影响因子：
11.1
作者：
Kazmirski, SL;Zhao, YX;Kuriyan, J
通讯作者：
Kuriyan, J