CONTROL OF DNA REPLICATION

DNA 复制的控制

• 批准号: 6289345
• 负责人: DHRUBA K CHATTORAJ
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6289345
• 关键词: DNA binding protein; DNA replication; DNA replication origin; Escherichia coli; cell cycle; gene expression; gene induction /repression; genetic promoter element; genetic regulatory element; nucleic acid repetitive sequence; plasmids; replicon; transcription factor

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.

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，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，我们在确定重复序列通过滴定启动子蛋白以及连接复制起始点来控制复制方面取得了重大进展。这些研究还表明，质粒范式适用于细菌复制。复制频率的调节P1质粒源(ORI<I/>)有5个结合位点(迭代)与质粒编码的启动子REPA相结合。已经有人提出，迭代通过滴定REPA或REPA介导的起始点的偶联来控制复制频率，从而导致空间位阻起始点的活性。滴定和耦合的影响都随着原液浓度的增加而增加。为了解决偶联的作用，我们开发了一种分析方法，包括比较其他相同基因的质粒单体和二聚体的拷贝数。我们的前提是，当两个起始点都在顺式结构中时，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结构中时更容易发生通信(耦合)，比如在二聚体中。与支持偶联模型的单体相比，二聚体拷贝数减少了两倍以上。使用拓扑分析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负向控制复制频率的有效机制。该方法可用于任何具有DNA环活性的蛋白质，如转录因子。复制诱导REPA<I/>基因转录<b/>我们发现REPA的转录是通过复制来激活的。启动子映射在迭代内，与其结合的REPA几乎完全抑制启动子的活性(自动抑制)。复制分叉的通过明显清除了结合的REPA的启动子，并为最大限度地表达REPA<I/>提供了机会之窗。相反，通过额外的迭代进行REPA滴定，不能有效地释放自抑制。在两倍额外迭代的存在下，拷贝数减少，但当额外的REPA以反式形式从构成源提供时，拷贝数可以恢复。这些结果表明，在一个滴定不能有效诱导启动子合成的系统中，可能需要复制诱导转录来确保启动子的可用性。DNA链开放：Dna结合位点位置的重要性链开放是启动DNA复制的关键步骤。由于DNA复制通常在起始阶段被控制，我们的前提是导致起点开放的步骤对于控制复制是重要的。在质粒P1中打开原点需要宿主启动子DNAA、DNA结构蛋白HU和REPA的参与。在大肠杆菌ORIC<i/>和包括P1在内的几个质粒上，Dna A蛋白都有特定的结合部位，Dna A盒，但对两个来源的Dna A盒的要求是不同的。虽然ORIC<i/>需要在不变位置上有多个框，但对于原始函数，在P1ori的两端都有一个共识框就足够了。通过KMnO4的探测，我们发现P1ori<i/>中链的打开效率取决于Dna A盒的位置和数量，但无论它们的位置如何，打开的位置都是相同的。复制的方向性也保持不变，这意味着Dna A可以从P1ori的两端起类似的作用。然而，OrI<I/>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。DNAA似乎正在与引发复合体接触，接触效率决定着拷贝数。因此，这种情况可能类似于ORIC<i/>，其中框对起始复合体的结构起关键作用，而不仅仅是增加蛋白质的局部浓度。结构蛋白的位点特异性结合Hu除了在特定位点与P1ori结合的两个启动子DNAA和REPA外，起点开放需要HU，它通常被认为是一种非特异性DNA结合蛋白。最近来自无关系统的研究表明，HU通过位点特异性结合帮助形成更高级别的核蛋白结构。我们发现，与非特异性DNA相比，HU与P1ori有更高的亲和力，提示HU可能与P1ori<I/>结合。通过体内足迹研究，已经获得了位点特异性结合的证据。目前，结合研究正在进行中。结合DNAA和REPA结合位点的知识，我们有可能理解允许链开放的起源拓扑。质粒复制频率的定量模型我们发展了一个基于单一函数(即转移函数)的低拷贝数质粒复制的随机模型，该模型描述了与细胞年龄有关的质粒复制的概率。这个函数可以直接从实验数据中推导出来。该模型现在正在扩展，以包括我们最近了解到的分子水平上的一些细节。这个项目是与美国国立卫生研究院NCRR的理论家保罗·莫里森合作完成的。