Control of DNA Replication

我们感兴趣的是了解DNA复制频率在细胞周期中是如何调节的。我们的系统是质粒P1，其复制子属于在细菌质粒中发现的复制子家族。这些复制子中的复制频率由短DNA重复序列(迭代)控制。在过去的一年里，我们在建立迭代子通过限制启动子蛋白以及通过结合的启动子连接(“手铐”)复制起点来控制复制方面取得了重大进展。这些研究还表明，质粒范例适用于细菌复制子。 复制频率的调节 P1质粒源有5个迭代子，与质粒编码的启动子REPA结合。有人提出，迭代子通过限制REPA或通过限制导致空间位阻的起始点的起始点来控制复制频率。为了解决手铐模型，我们开发了一种分析方法，包括比较携带质粒单体或同基因质粒二聚体的细胞的质粒拷贝数。我们的前提是，当两个起始点都在顺式，如二聚体中时，由于一个位置在另一个位置附近的局部浓度比它们在反式位置时，如在单体中，通信(手铐)更容易发生。与支持手铐模型的单体相比，二聚体拷贝数减少了四倍。使用一种新的检测方法也在体内获得了起源之间直接物理相互作用的证据。我们的研究首次提供了生理学证据，证明起始点耦合可以是减少复制的有效机制。这里开发的分析方法可以应用于任何具有DNA环化活性的蛋白质，例如转录因子。 增加质粒拷贝数的启动子突变体 为了建立手铐模型，我们分离了能增加P1质粒拷贝数的启动子突变体。这些突变体显然在控制质粒过度复制方面存在缺陷，正在进行体内和体外的手铐测试。拷贝数的增加与手铐缺陷之间的强烈相关性将确立手铐在拷贝数控制中的重要性。如果只在突变体的子集中发现了手铐缺陷，那么就可以证明存在其他控制模式。我们还在开发新的检测方法，可以独立处理手铐问题。 Dna A盒在P1质粒起源中的作用 Dna A蛋白是在多种细菌和质粒复制体中启动DNA复制所必需的。这些复制子中的复制起点总是包含蛋白质的特定结合位点，称为Dna A盒。质粒P1在其起始点的两端各含有一组DNAA盒，但可以与其中任何一组一起工作。我们发现，无论框是在起点的两端还是在起点的一端，起始点的位置、复制叉子的起始点和复制的方向性都不会改变。复制在所有情况下都是双向的。这些结果表明，DNAA在起始点的两端具有相似的功能。尽管有这种戏剧性的灵活性，但原点两端的盒子位置(相位)的微小变化显著地改变了原点的效率。最有可能的是，这些盒子不仅有助于增加局部DNAA的浓度，而且允许蛋白质以一种分阶段敏感的方式接触起始复合体的其他成分。 一个偶然的观察到，与起始点无关的序列会极大地影响起始点的活性。在外源序列中发现了IHF位点，IHF蛋白被发现是复制所必需的。我们目前正在研究IHF激活P1起源的机制。在各种DNA交易中，IHF允许与远距离结合的蛋白质之间的相互作用。我们正在追求一种模型，在该模型中，IHF通过将远端DNA引入起始复合体附近来激活复制。末端DNA与启动子的非特异性结合足以稳定该复合体。DNA弯曲激活转录也有类似的机制。 组蛋白样蛋白Hu的位点特异性结合 除了在特定位点与P1起始部位结合的两个启动子DNAA和REPA外，启动还需要HU。这种蛋白通常被认为是一种非特异的DNA结合蛋白。最近对大肠杆菌Gal启动子和噬菌体Mu转座的研究表明，Hu通过位点特异性结合帮助形成更高顺序的核蛋白结构。我们发现，与非特异性DNA相比，HU与P1起始点具有更高的亲和力，提示HU可能与P1起始点特异结合。体内足迹研究与Hu在含有迭代子的区域的特异性结合一致。在体外，胡舒立也在迭代粒子上显示了特定的足迹，但其作用弱于体内。在体外的超螺旋模板中，HU抑制转录的浓度范围与抑制GAL启动子所需的浓度范围相同。HU的位点特异性结合如何有助于启动仍有待了解。

Our interest is to understand how the DNA replication frequency is adjusted in the cell cycle. Our system is plasmid P1 whose replicon belongs to a family of replicons found in bacterial plasmids. The replication frequency in these replicons is controlled by short DNA repeat sequences (iterons). In the past year we have made significant progress in establishing that the iterons control replication by limiting initiator protein as well as by coupling ("handcuffing") replication origins via the bound initiators. These studies also showed that the plasmid paradigm is applicable to bacterial replicons. Regulation of Replication Frequency The P1 plasmid origin has five iterons that bind the plasmid-encoded initiator, RepA. It has been proposed that iterons control replication frequency by either limiting RepA or by handcuffing origins that causes steric hindrance to origin activity. To address the handcuffing model, we have developed an assay that involves comparison of plasmid copy numbers from cells that carry either plasmid monomer or isogenic plasmid dimer. Our premise is that communication (handcuffing) would occur more readily when the two origins are in cis, as in a dimer, because of higher local concentration of one site in the vicinity of another, than when they are in trans as in monomers. Dimer copy number was four-fold lower as compared to monomer in support of the handcuffing model. Evidence for direct physical interactions between origins was also obtained in vivo using a novel assay. Our studies provide the first physiological evidence that origin coupling can be an effective mechanism to reduce replication. The assays developed here can be applied to any protein, such as a transcription factor, with DNA looping activity. Initiator Mutants that Increase Plasmid Copy Number To establish the handcuffing model, we have isolated initiator mutants that increase the P1 plasmid copy number. These mutants, apparently defective in controlling plasmid overreplication, are being tested for handcuffing in vivo and in vitro. A strong correlation between copy number increase and the handcuffing defect will establish the importance of handcuffing in copy number control. If the handcuffing defect is found only in a subset of mutants, then presence of other modes of control can be argued. We are also developing new assays that can address handcuffing independently. Role of DnaA Boxes in the P1 Plasmid Origin The DnaA protein is essential for initiation of DNA replication in a wide variety of bacterial and plasmid replicons. The replication origin in these replicons invariably contains specific binding sites for the protein, called DnaA boxes. Plasmid P1 contains a set of DnaA boxes at each end of its origin but can function with either one of the sets. We found that the location of origin-opening, initiation site of replication forks, and directionality of replication do not change whether the boxes are present at both or at one of the ends of the origin. Replication was bidirectional in all cases. These results imply that DnaA functions similarly from the two ends of the origin. In spite of this dramatic flexibility, small changes in the box position (phasing) at either end of the origin significantly altered the origin efficiency. Most likely, the boxes not only help to increase local DnaA concentration but allow the protein to contact other components of the initiation complex in a phasing sensitive manner. A serendipitous observation was that sequences extraneous to the origin could greatly influence the origin activity. An IHF site was found in the extraneous sequence and the IHF protein was found to be essential for replication. We are currently studying the mechanism by which IHF could be activating the P1 origin. In a variety of DNA transactions IHF allows interactions between proteins bound to distant sites. We are pursuing a model where IHF is activating replication by bringing in distal DNA in close proximity of the initiation complex. The non-specific binding of the distal DNA to initiators could suffice to stabilize the complex. A similar mechanism has been suggested for the activation of transcription by DNA bending. Site-specific Binding of the Histone-like Protein, HU In addition to two initiators, DnaA and RepA, that bind to P1 origin at specific sites, initiation also requires HU. This protein is generally known to be a non-specific DNA binding protein. Recent studies in E. coli gal promoter and in phage Mu transposition have indicated that HU helps to form higher order nucleoprotein structures by site-specific binding. We have found that HU has higher affinity for the P1 origin compared to nonspecific DNA suggesting that HU may bind to P1 origin site-specifically. Footprinting studies in vivo were consistent with site-specific binding of HU in the region containing the iterons. HU also showed footprints specifically on iterons in vitro but the effect was weaker than in vivo. In supercoiled templates in vitro, HU repressed transcription in the same concentration range as was required to repress the gal promoter. How the site-specific binding of HU could help initiation remains to be understood.