Control of DNA Replication

我们的兴趣是了解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，它属于细菌质粒中常见的复制子家族，其复制频率由短重复DNA序列控制。在过去的一年里，我们已经取得了重大进展，建立了重复控制复制的滴定起始蛋白，以及通过耦合复制起点。这些研究也表明，质粒范式适用于细菌复制子。复制频率的调节P1质粒起点（ori）有5个结合位点（iteron），用于质粒编码的起始子RepA。有人提出，iteron控制复制频率通过滴定RepA或RepA介导的耦合的起源，导致空间位阻的起源活动。滴定和偶联的影响预计将增加与源浓度的增加。为了说明偶联的作用，我们开发了一种检测方法，该方法涉及比较质粒单体和二聚体的拷贝数，否则它们是等基因的。我们的前提是，通信（耦合）将更容易发生时，两个起源是顺式，如在一个二聚体，因为更高的局部浓度的一个网站附近的另一个，比当他们在反式单体。二聚体拷贝数比单体低两倍以上，支持偶联模型。证据来源之间的直接物理相互作用也获得了在体内使用拓扑分析。我们的研究提供了第一个生理学证据，起点偶联可以是一个有效的负控制复制频率的机制。本发明的检测方法可应用于任何具有DNA成环活性的蛋白质，如转录因子。repA基因的复制诱导转录我们发现repA基因的转录是由复制激活的。启动子映射在iteron和RepA结合到他们几乎完全抑制启动子活性（自动阻遏）。复制叉的通过显然清除了结合的RepA的启动子，并为最大repA表达提供了机会窗口。与此相反，自动阻遏没有有效地释放后，RepA滴定额外的iterons。在存在的两倍额外的iterons，拷贝数减少，但它可以重新获得时，额外的RepA提供反式从组成性来源。这些结果表明，RepA是不是在过量，复制诱导的转录可能需要，以确保启动子的可用性在一个系统中，引发剂的合成是不是有效地诱导titration.DNA链开放：DnaA结合位点PositionStrand开放的重要性是在启动DNA复制的关键步骤。由于DNA复制通常在起始阶段就受到控制，因此我们的前提是，导致起点开放的步骤对于控制复制很重要。质粒P1中的起点开放需要宿主起始子DnaA、DNA结构蛋白HU和RepA的参与。DnaA蛋白在大肠杆菌的起源中具有特异的结合位点，即DnaA盒。coli、oriC和包括P1在内的几种质粒，但这两种来源对DnaA盒的要求不同。而oriC需要在不变的位置多个框，一个单一的共识框在P1ori的任何一端就足以为原点功能。通过KMnO4探针分析，我们发现P1ori的链开放效率依赖于DnaA盒的配置和数量，但无论其配置如何，开放的位置保持不变。复制的方向性也保持不变，这意味着DnaA可以从P1ori的两端类似地发挥作用。然而，在ori两端的盒子位置的微小变化显着降低了打开的效率和质粒拷贝数。似乎DnaA与起始复合物接触，接触效率决定了拷贝数。这种情况，因此，可能是类似的oriC，其中的框关键有助于架构的起始复合物，而不仅仅是增加当地的蛋白质的浓度。位点特异性结合的建筑蛋白，HU除了两个引发剂，DnaA和RepA，结合到P1ori在特定的网站，起源开放需要HU，这是众所周知的是一个非特异性的DNA结合蛋白。最近对非相关系统的研究表明，HU通过位点特异性结合帮助形成更高级的核蛋白结构。我们已经发现，HU具有更高的亲和力P1ori相比，非特异性DNA表明，HU可能结合P1ori位点特异性。位点特异性结合的证据已通过体内足迹法研究获得。目前，结合研究正在体外进行。结合DnaA和RepA的结合位点的知识，它可以理解的起源拓扑结构，允许链开放。质粒复制频率的定量模型我们已经开发了一个随机模型的低拷贝数质粒复制的基础上，一个单一的功能，描述质粒复制的概率与细胞年龄（即过渡函数）。该函数可直接从实验数据导出。该模型现在正在扩展，以包括我们最近了解到的分子水平上的一些细节。这个项目是与美国国立卫生研究院国家癌症研究中心的理论家保罗莫里森合作的成果。

Our interest is to understand how the DNA replication frequency is adjusted in the cell cycle. Our system is plasmid P1 that belongs to a family of replicons commonly found in bacterial plasmids whose replication frequency is controlled by short repeating DNA sequences. In the past year we have made significant progress in establishing that the repeats control replication by titrating initiator protein as well as by coupling replication origins. These studies also show that the plasmid paradigm is applicable to bacterial replicons.Regulation of Replication Frequency The P1 plasmid origin (ori<i/>) has five binding sites (iterons) for the plasmid-encoded initiator, RepA. It has been proposed that iterons control replication frequency by either titrating RepA or RepA-mediated coupling of origins which causes steric hindrance to origin activity. The effects of both titration and coupling are expected to increase with increase of origin concentration. To address the role of coupling, we have developed an assay that involves comparison of copy numbers of plasmid monomer and dimer that are otherwise isogenic. Our premise is that communication (coupling) 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 more than two-fold lower as compared to monomer in support of the coupling model. Evidence for direct physical interactions between origins was also obtained in vivo using a topological assay. Our studies provide the first physiological evidence that origin coupling can be an effective mechanism for negative control of the replication frequency. The assay developed here can be applied to any protein, such as a transcription factor, with DNA looping activity.Replication-induced Transcription of the repA<i/> Gene<B/> We have found that transcription of repA is activated by replication. The promoter maps within the iterons and RepA binding to them represses the promoter activity almost totally (autorepression). The passage of the replication fork apparently cleans the promoter of bound RepA and provides a window of opportunity for maximal repA<i/> expression. In contrast, autorepression was not efficiently released upon RepA titration by extra iterons. In the presence of two-fold extra iterons, the copy number reduced but it could be regained when extra RepA was supplied in trans from a constitutive source. These results argue that RepA is not made in excess and, replication-induced transcription may be required to ensure initiator availability in a system where initiator synthesis is not efficiently induced by titration.DNA Strand Opening: Importance of DnaA Binding Site PositionStrand opening is a crucial step in the initiation of DNA replication. Since DNA replication is normally controlled at the stage of initiation, our premise is that steps leading to origin opening are important for controlling replication. Origin opening in plasmid P1 requires participation of host initiator DnaA, a DNA architectural protein HU, and RepA. The DnaA protein has specific binding sites, the DnaA boxes, in the origin of E. coli, oriC<i/>, and of several plasmids including P1, but the requirements of DnaA boxes are different for the two origins. Whereas oriC<i/> requires multiple boxes at invariant positions, a single consensus box at either end of the P1ori<i/> suffices for the origin function. By probing with KMnO4, we found that the efficiency of strand opening in P1ori<i/> depended on the disposition and number of DnaA boxes, but the location of opening remained the same regardless of their disposition. The directionality of replication also remained the same implying that DnaA can function similarly from either end of P1ori<i/>. However, small changes in box positions at either end of the ori<i/> reduced the efficiency of opening and plasmid copy number significantly. It appears that DnaA is contacting the initiation complex and the contact efficiency is determining the copy number. The situation, therefore, could be similar to oriC<i/>, where the boxes critically contribute to the architecture of the initiation complex and not merely increase the local concentration of the protein.Site-specific Binding of the Architectural Protein, HUIn addition to two initiators, DnaA and RepA, that bind to P1ori at specific sites, origin opening requires HU, which is generally known to be a non-specific DNA binding protein. Recent studies from unrelated systems have indicated that HU helps to form higher order nucleoprotein structures by site-specific binding. We have found that HU has higher affinity for P1ori compared to nonspecific DNA suggesting that HU may bind to P1ori<i/> site-specifically. The evidence for site-specific binding has been obtained by in vivo footprinting studies. Presently, the binding studies are being conducted in vitro. Together with the knowledge of binding sites for DnaA and RepA it may be possible to understand the origin topology that allows strand-opening.A Quantitative Model of Plasmid Replication FrequencyWe have developed a stochastic model of low-copy-number plasmid replication based on a single function describing the probability of plasmid replication with cell age (i.e. a transition function). This function can be derived directly from experimental data. The model is now being expanded to include some of the details at the molecular level that we have learnt more recently. This project is a collaborative effort with a theoretician, Paul Morrison, of NCRR, NIH.