Interaction of the sliding clamp β-subunit and Hda, a DnaA-related protein
Interaction of the sliding clamp β-subunit and Hda, a DnaA-related protein
复制标题
DOI：
10.1128/jb.186.11.3508-3515.2004
复制
发表时间：
2004-06-01
影响因子：
3.2
通讯作者：
Kongsuwan, K
中科院分区：
文献类型：
--
作者：
Kurz, M;Dalrymple, B;Kongsuwan, K
在大肠杆菌中，复制起始蛋白DnaA、DNA聚合酶III的β亚基（滑动钳蛋白）和Hda（最近发现的DNA相关蛋白）之间需要相互作用，才能通过ATP的加速水解将活性的ATP结合形式的DnaA转化为非活性的adp结合形式。这种ATP的快速水解被认为是细胞周期中阻止多次起始的主要机制，并作为从起始到复制的分子开关。然而，DNA合成中这一关键步骤的生化机制尚未得到解决。利用纯化的Hda和β蛋白进行平板结合实验和Ni-nitrilotriacetic acid pull - down分析，我们首次在体外发现Hda与β蛋白直接相互作用。在Hda蛋白的氨基端发现了一个新的β结合基序，与先前鉴定的β结合五肽基序（QL[SD]LF）相关，其一致序列为QL[SP]LPL。在六肽基序中发生氨基酸变化的Hda突变体，其结合β的能力严重缺陷。一个含有大肠杆菌Hda- β结合基序的10氨基酸肽在Hda- β相互作用实验中被证明与Hda竞争结合。这些结果表明Hda与β的相互作用是通过六肽序列介导的。我们认为，这种相互作用可能对导致dna失活和防止过度启动复制的事件至关重要。
In Escherichia coli, interactions between the replication initiation protein DnaA, the beta subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and beta proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with beta in vitro. A new beta-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified beta-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind beta. A 10-amino-acid peptide containing the E. coli Hda beta-binding motif was shown to compete with Hda for binding to beta in an Hda-beta interaction assay. These results establish that the interaction of Hda with beta is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.