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整的。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。在过去的一年中，我们在确定重复序列通过滴定启动蛋白以及偶联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，质粒模式适用于细菌复制子。P1质粒起源（ori<i/>）有5个与质粒编码的启动物RepA结合的位点（iterons）。有人提出，迭代子通过滴定RepA或RepA介导的起点偶联来控制复制频率，从而导致起点活性的位阻。随着初始浓度的增加，滴定和偶联的作用都将增强。为了解决偶联的作用，我们开发了一种检测方法，包括比较质粒单体和二聚体的拷贝数，否则是等基因的。我们的前提是，当两个起源为顺式时，如在二聚体中，因为一个位点在另一个位点附近的局部浓度较高，而当它们为反式时，如在单体中，通讯（偶联）更容易发生。二聚体的拷贝数比单体低2倍以上，支持耦合模型。起源之间的直接物理相互作用的证据也通过拓扑分析在体内获得。我们的研究提供了第一个生理证据，证明起源耦合可以是负控制复制频率的有效机制。这里开发的试验可以应用于任何蛋白质，如转录因子，具有DNA环活性。复制诱导的repA<i/>基因的转录<B/>我们发现repA的转录是由复制激活的。启动子在迭代子内映射，与它们结合的RepA几乎完全抑制启动子的活性（自抑制）。复制叉的通过明显地清除了结合RepA的启动子，并为RepA <i/>的最大表达提供了机会。相反，通过额外的干扰素进行RepA滴定时，自抑制不能有效地释放。当存在两个额外的迭代子时，拷贝数减少，但当从组成源以trans形式提供额外的RepA时，拷贝数可以恢复。这些结果表明，RepA不会过量产生，并且在滴定不能有效诱导引发剂合成的系统中，可能需要复制诱导转录来确保引发剂的可用性。DNA链打开：DNA结合位点位置的重要性链打开是DNA复制起始的关键步骤。由于DNA复制通常在起始阶段受到控制，我们的前提是导致起点打开的步骤对控制复制很重要。质粒P1的起始打开需要宿主启动物DnaA、DNA构建蛋白HU和RepA的参与。在大肠杆菌、oriC<i/>和包括P1在内的几种质粒中，DnaA蛋白具有特定的结合位点，即DnaA盒子，但两种来源对DnaA盒子的要求不同。oriC<i/>需要在不变位置有多个盒子，而在P1ori<i/>的任意一端有一个共识盒子就足以满足原点函数。通过KMnO4探针，我们发现P1ori<i/>的链打开效率取决于dna盒的配置和数量，但无论它们的配置如何，打开的位置保持不变。复制的方向性也保持不变，这意味着dna可以从P1ori<i/>的任何一端起类似的作用。然而，在序列<i/>的两端，盒子位置的微小变化显著降低了打开效率和质粒拷贝数。dna与起始复合体接触，接触效率决定了dna的拷贝数。因此，这种情况可能类似于oriC<i/>，其中盒子对起始复合物的结构至关重要，而不仅仅是增加蛋白质的局部浓度。除了DNA和RepA两种启动子在特定位点与P1ori结合外，起始点打开还需要HU，它通常被认为是一种非特异性DNA结合蛋白。最近来自不相关系统的研究表明，HU通过位点特异性结合有助于形成高阶核蛋白结构。我们发现与非特异性DNA相比，HU对P1ori具有更高的亲和力，这表明HU可能特异性地结合P1ori<i/>位点。位点特异性结合的证据是通过体内足迹研究获得的。目前，结合研究正在体外进行。结合dna和RepA结合位点的知识，有可能理解允许链打开的起源拓扑结构。质粒复制频率的定量模型我们建立了一个低拷贝数质粒复制的随机模型，该模型基于描述质粒复制随细胞年龄的概率的单一函数（即过渡函数）。这个函数可以直接从实验数据推导出来。该模型现在正在扩展，以包括我们最近了解到的分子水平的一些细节。这个项目是与美国国立卫生研究院NCRR的一位理论家Paul Morrison合作完成的。

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.