Mechanisms of DNA replication elongation

DNA复制的第一步之一是将复制蛋白装载到要复制的DNA上。T4基因59蛋白与DNA结合，并在此过程中发挥关键作用，通过极大地刺激T4基因41解旋酶在复制部位的负载，导致形成有效复制T4基因组所需的完整蛋白质复合体。人们认为在所有生物体的DNA复制启动过程中都会发生类似的事件，因此通过研究携带蛋白质的基因59的生物化学获得的洞察力可能有助于阐明DNA复制的一般机制。有效的T4DNA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起始处的过程复制体组装过程中的相互作用。在一个模型中，32个SSB和59个蛋白质形成一个复合体，将复制的41解旋酶招募到起始点。在第二种模式中，32SSB必须首先被辅助解旋酶去除起始点或被59蛋白取代，然后才能装载41和组装复制体。我们使用一组59个突变体和32个截断来直接在体外测试这些模型，并确定32个SSB抑制59介导的解旋酶在设计用于模拟起源的分叉DNA底物上的加载。这种抑制作用在32-B和32-A中都观察到，32-B是一种不结合DNA的32SSB截短，32-A保留了DNA结合活性。此外，缺乏32个相互作用的59个突变体不受32-B的影响，而是受32-A的影响。因此，32SSB可能通过两种不同的机制抑制59介导的解旋酶负载，即竞争分叉DNA和直接与59在溶液中相互作用。净效应是32个SSB破坏了59蛋白和41解旋酶在分叉DNA上形成的复合体，这意味着这三种蛋白质之间没有负载能力的三元复合体。然而，32SSB和59蛋白之间的复合体在T4DNA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中，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的突变破坏，T4DNA复制就会中断，并合成异常大的滞后链片段。我们的结果表明，59在T4DNA复制过程中至少有两个功能，促进了41解旋酶在起始处的装载，并在复制体沿染色体移动时组织了滞后的链合成。 虽然T4-59蛋白被认为是在病毒复制起始处的复制体组装过程中主动装载复制的41解旋酶，但一些T4起始蛋白在缺乏59蛋白的情况下在感染过程中是活跃的，这表明59蛋白对于解旋酶的装载并不是绝对必要的。为了准确地确定正常的T4复制所必需的生化活性，我们研究了几个已定义的59个突变体在感染期间对体外复制和病毒DNA合成的影响。正如预期的那样，DNA结合缺陷的59突变体在体外和感染期间都不能刺激解旋酶负载和T4DNA复制。然而，缺乏解旋酶相互作用的59个突变体不能有效地装载41个解旋酶，也不能在体外刺激41个依赖的DNA复制，在感染过程中对病毒复制几乎没有明显的影响。在感染过程中合成的DNA总量和T4染色体上的复制模式都与正常感染非常相似。用这个59突变体观察到的复制几乎完全依赖于T4dda解旋酶，这意味着这个辅助解旋酶参与了41解旋酶的加载。另有59个突变株与32个单链DNA结合蛋白(SSB)相互作用不足，对T4复制有不同的影响。该突变导致总DNA合成滞后，无论是在体外还是在感染期间，复制模式都没有接近起始点的典型DNA合成高峰。起源合成的减少显然是由59个把关活性的缺陷引起的，该缺陷将T4DNA聚合酶保持在原位，直到41个解旋酶被加载到起源上。然而，解旋酶相互作用缺失的59个突变株在32个SSB覆盖的底物上具有正常的门控活性，而在32个SSB相互作用缺失的59个突变株则不具有正常的门控活性。因此，59蛋白在感染过程中的主要功能似乎是针对T4起始处的复制体组装。

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.