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首页> 外文期刊>Biochemistry >Interaction between the T4 helicase-loading protein (gp59) and the DNA polymerase (gp43): A locking mechanism to delay replication during replisome assembly
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Interaction between the T4 helicase-loading protein (gp59) and the DNA polymerase (gp43): A locking mechanism to delay replication during replisome assembly

机译：T4解旋酶负载蛋白（gp59）与DNA聚合酶（gp43）之间的相互作用：锁定机制以延迟复制体组装过程中的复制。

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摘要

The T4 helicase-loading protein (gp59) has been proposed to coordinate leading- and laggingstrand DNA synthesis by blocking leading-strand synthesis during the primosome assembly. In this work, we unambiguously demonstrate through a series of biochemical and biophysical experiments, including single-molecule fluorescence microscopy, that the inhibition of leading-strand holoenzyme progression by gp59 is the result of a complex formed between gp59 and leading-strand polymerase (gp43) on DNA that is instrumental in preventing premature replication during the assembly of the T4 replisome. We find that both the polymerization and 3' --> 5' exonuclease activities of gp43 are totally inhibited within this complex. Chemical cross-linking of the complex followed by tryptic digestion and peptide identification through matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry identified Cys 169 of gp43 and Cys215 of gp59 as residues in a region of a protein-protein contact. With the available crystal structures for both gp43 and gp59, a model of the complex was constructed based on shape complementarity, revealing that parts of the C-terminal domain from gp59 insert into the interface created by the thumb and exonuclease domains of gp43. This insertion effectively locks the polymerase into a conformation where switching between the pol and editing modes is prevented. Thus, continued assembly of the replisome through addition of the primosome components and elements of the laggingstrand holoenzyrne can occur without leading-strand DNA replication.
机译：T4解旋酶加载蛋白（gp59）已被提出通过在原核小体装配过程中阻断前导链合成来协调前导链和后链DNA的合成。在这项工作中，我们通过一系列生物化学和生物物理实验（包括单分子荧光显微镜）明确证明，gp59对前导全酶的抑制是gp59与前导聚合酶（gp43 ）有助于防止T4复制体装配过程中过早复制的DNA。我们发现，在该复合物中，gp43的聚合和3'-> 5'核酸外切酶活性均被完全抑制。配合物的化学交联，然后通过基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱进行胰蛋白酶消化和肽鉴定，鉴定出gp43的Cys 169和gp59的Cys215为残基。蛋白质-蛋白质接触。利用gp43和gp59的可用晶体结构，基于形状互补性构建了复合物模型，揭示了来自gp59的C末端结构域的部分插入到gp43的拇指和核酸外切酶结构域创建的界面中。这种插入有效地将聚合酶锁定为构象，从而防止了pol和编辑模式之间的切换。因此，通过添加落后链甲氧苄基环的原核小体成分和元件，可以继续组装复制体而无需前导链DNA复制。

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