The role of recombination in hairpin telomere maintenance.

伯氏疏螺旋体是一种成熟的研究DNA发夹端粒的自然科学模型系统。发夹端粒是复制线性DNA问题的不同寻常的解决方案。发夹端粒是位于线形染色体或质粒末端的闭合发夹。它们消除了通常存在于线性DNA中的自由端，从而消除了复制问题，该问题会导致每次细胞分裂时DNA末端的信息逐渐丢失。线性染色体中的DNA复制是双向的，从DNA的中间开始，向两个HP端粒进行。围绕hp端粒的复制将产生通过复制的端粒(Rtel)连接在一起的环形二聚体子体DNA。为了将子体DNA在子体细胞之间分裂，环状二聚体由一种特殊的酶--REST处理。REST在Rtel连接处进行DNA切割和重新连接反应，该连接分离线形DNA子体DNA，在线形DNA末端再生新的HP端粒。我的实验室研究REST的性质。在这项提案中，我们将检验/挑战上述模型的假设，即rtel连接总是由正常的DNA复制功能产生，在hp端粒周围的DNA合成的延续。在实验室的最新进展中，我们发现REST具有意想不到的生化特性，使其能够分离DNA链并重新退火。我们还发现，REST与一种蛋白质有关，这种蛋白质促进了相同DNA副本之间的遗传信息交换。我们将这个蛋白命名为RARA(代表REST相关重组酶A)。这些活动与许多生物体中涉及的蛋白质非常相似，修复DNA损伤和修复DNA损伤部位停滞不前的DNA复制复合体。*我们假设，这些与修复酶的相似性表明，hp端粒在细胞中看起来像DNA损伤，REST/RARA提供的功能是完成疏螺旋体线性DNA复制所必需的。我们将通过观察试管中和实验室培养的疏螺旋体中蛋白质的性质来检验我们的假设。我们将在试管中将REST和RARA的性质与已知的修复酶进行比较，以确定与已知修复功能的相似程度。然后，我们将干扰这些蛋白质在细胞中的功能，看看这是否会干扰细胞复制DNA的能力。如果这些蛋白质对细胞存活具有重要的DNA修复类功能，那么它们将构成疏螺旋体中这些修复功能的第一个被识别的例子。此前人们认为疏螺旋体缺乏这些功能。对复制酵母和哺乳动物细胞线性DNA所涉及的类似基本问题的研究，有助于我们在理解细胞转化和衰老等基本生物学问题方面取得巨大进展。

Borrelia burgdorferi, is a well-established natural science model system for the study of DNA hairpin (hp) telomeres. Hairpin telomeres are an unusual solution to the problem of copying linear DNAs. Hairpin telomeres are closed hairpins at linear chromosome or plasmid termini. They eliminate the free end normally present in linear DNAs and, thereby, eliminate the copying problem that would result in the progressive loss of information at the DNA ends every time the cells divide. DNA copying in the linear chromosome is bidirectional and starts in the middle of the DNA and proceeds towards the two hp telomeres. Replication, around the hp telomeres, would produce circular dimer daughter DNAs linked together via replicated telomere (rTel) junctions. In order for the daughter DNAs to be divided between the daughter cells the circular dimer is processed by a specialized enzyme, ResT. ResT performs DNA cleavage and rejoining reactions at the rTel junctions that separates linear daughter DNAs regenerating new hp telomeres at the linear DNA ends.******My lab studies the properties of ResT. In this proposal we will examine/challenge the assumption of the above model that rTel junctions are always produced by normal DNA copying functions, in a continuation of DNA synthesis around the hp telomeres. In recent progress in the lab we have discovered that ResT has unexpected biochemical properties that allow it to separate DNA strands and re-anneal them. We have also discovered that ResT is associated with a protein that promotes exchange of genetic information between copies of the same DNA. We have named this protein RarA (for ResT-associated recombinase A). These activities are very similar to those of proteins involved, in many organisms, with the repair of DNA damage and the repair of stalled DNA copying complexes at sites of DNA damage.******We hypothesize that these similarities to repair enzymes indicate that the hp telomeres look to the cell like DNA damage and that functions provided by ResT/RarA are needed to complete the copying of Borrelia's linear DNAs. We will test our hypothesis by looking at the properties of the proteins in the test tube and in Borrelia grown in the lab. We will compare the properties of ResT and RarA to known repair enzymes in the test tube to determine the extent of the similarities with the known repair functions. Then we will interfere with the function of these proteins in the cell to see if this interrupts the cell's ability to copy their DNA. If these proteins have DNA repair-like functions important for cell survival then they would constitute the first identified examples of these repair functions in Borrelia. It was previously thought that Borrelia lacked these functions. Study of similar fundamental issues involved in copying the linear DNA of yeast and mammalian cells has contributed to huge advances in our understanding of fundamental biological issues like cellular transformation and aging.