Mechanisms of DNA replication elongation

DNA复制的第一步是将复制蛋白装载到待复制的DNA上。T4基因59蛋白结合DNA，在这一过程中发挥关键作用，通过极大地刺激T4基因41解旋酶在复制位点的负载，导致形成T4基因组有效复制所必需的完整蛋白复合物。类似的事件被认为发生在所有生物DNA复制的起始阶段，因此通过研究基因59装载蛋白的生物化学获得的见解可能有助于阐明涉及DNA复制的一般机制。有效的T4 DNA复制还需要另一种DNA结合蛋白，32单链结合蛋白（SSB）的功能。至少出现了两种模型来描述这两种蛋白质在T4起源的过程复制体组装过程中的相互作用。在一个模型中，32 SSB和59蛋白形成一个复合体，将复制解旋酶招募到原点。在第二种模型中，32 SSB必须首先被辅助解旋酶从起源上清除或被59蛋白置换，然后才能装载41并组装复制体。我们使用59个突变体和32个截短片段在体外直接测试了这些模型，并确定32个SSB抑制了59介导的解旋酶在分叉DNA底物上的装载。这种抑制作用在32- b（不结合DNA的32- SSB截断）和32- a（保留DNA结合活性）中都有观察到。此外，32个相互作用的59个突变体不受32- b的影响，但受32- a的影响。因此，32 SSB似乎可以通过两种不同的机制抑制59介导的解旋酶装载，即分叉DNA的竞争和溶液中与59的直接相互作用。净效应是32 SSB破坏了分叉DNA上59个蛋白质和41个解旋酶之间的形成复合物，这意味着在这三种蛋白质之间没有形成负载能力的三元复合物。然而，32 SSB和59蛋白之间的复合物在T4 DNA复制过程中确实具有功能意义。32可以将59蛋白招募到单链DNA上，这一活动需要与59蛋白直接相互作用。如果这种相互作用被59突变破坏，T4 DNA复制被破坏，并合成异常大的滞后链片段。我们的研究结果表明，在T4 DNA复制过程中，59至少有两个功能作用，促进41解旋酶在起源处的装载，并在复制体沿着染色体移动时组织滞后链的合成。

One of the first steps of DNA replication is the loading of the replication proteins onto the DNA that is to be replicated. The T4 gene 59 protein binds DNA and plays a key role in this process, by greatly stimulating the loading of T4 gene 41 helicase at the site of replication, leading to the formation of the complete protein complex necessary for efficient replication of the T4 genome. Similar events are thought to happen during the initiation of DNA replication in all organisms, so the insight gained through studying the biochemistry of gene 59 loading protein may help to illuminate general mechanisms involved in DNA replication. Efficient T4 DNA replication also requires the functions of another DNA binding protein, the 32 single-stranded binding protein (SSB). At least two models have emerged to describe the interactions between these two proteins during the assembly of processive replisomes at the T4 origins. In one model 32 SSB and 59 protein form a complex that recruits the replicative 41 helicase to the origins. In the second model, 32 SSB must first be wiped off the origins by an accessory helicase or displaced by 59 protein before 41 can be loaded and the replisome assembled. We directly tested these models in vitro using a panel of 59 mutants and 32 truncations and determined that 32 SSB inhibits 59 mediated helicase loading on forked DNA substrates designed to mimic origins. This inhibition was observed with both 32-B, a 32 SSB truncation that does not bind DNA, and 32-A, which retains DNA binding activity. Moreover, a 59 mutant deficient in 32 interactions is not affected by 32-B but is by 32-A. Thus, it appears that 32 SSB can inhibit 59 mediated helicase loading through two separate mechanisms, competition for fork DNA and direct interaction with 59 in solution. The net effect is that 32 SSB disrupts formation complexes between 59 protein and 41 helicase on forked DNA, implying that there is no loading competent, ternary complex formed between the three proteins. Yet, complexes between 32 SSB and 59 protein do have a functional significance during T4 DNA replication. 32 can recruit 59 protein to single-stranded DNA, an activity that requires direct interaction with 59 protein. If this interaction is disrupted by mutation of 59, T4 DNA replication is disrupted, and abnormally large lagging strand fragments are synthesized. Our results suggest that 59 has at least two functional roles during T4 DNA replication, facilitating 41 helicase loading at the origins and organizing lagging strand synthesis as the replisome moves along the chromosome. Although T4 59 protein is thought to actively load the replicative 41 helicase during replisome assembly at the viral origins of replication, some T4 origins are active during infection in the absence of 59 protein, This indicates that 59 protein is not absolutely necessary for helicase loading. To determine exactly what biochemical activities are necessary for normal T4 replication we investigated the effects of several defined 59 mutants on in vitro replication and viral DNA synthesis during infection. As expected, a 59 mutant deficient in DNA binding was incapable of stimulating helicase loading and T4 DNA replication, both in vitro and during infection. Yet, the 59 mutant deficient in helicase interaction, which was unable to efficiently load 41 helicase or stimulate 41 dependent DNA replication in vitro, had little obvious effect on viral replication during infection. Both the total amount of DNA synthesized over the course of infection and the pattern of replication across the T4 chromosome were very similar to normal infections. The replication observed with this 59 mutant was almost entirely dependent on T4 dda helicase, implying that this accessory helicase is involved in 41 helicase loading. Another 59 mutant deficient in interactions with 32 single-stranded DNA binding protein (SSB) had a different effect on T4 replication. This mutant caused a lag in total DNA synthesis, both in vitro and during infection, and the pattern of replication was devoid of the typical peaks of DNA synthesis near the origins. The reduction in origin synthesis is apparently caused by a defect in 59 gatekeeping activity, holding T4 DNA polymerase in place until 41 helicase is loaded onto the origins. Whereas the 59 mutant deficient in helicase interactions has normal gatekeeping activity on 32 SSB coated substrates, the 59 mutant deficient in 32 SSB interactions does not. Hence, it appears that the primary function of 59 protein during infection is to target replisome assembly to the T4 origins.