The role of recombination in hairpin telomere maintenance.

伯氏疏螺旋体是研究DNA发夹（hp）端粒的一个成熟的自然科学模型系统。发夹端粒是解决线性dna复制问题的一种不同寻常的方法。发夹端粒是线性染色体或质粒末端闭合的发夹。它们消除了线性DNA中通常存在的自由末端，从而消除了每次细胞分裂时导致DNA末端信息逐渐丢失的复制问题。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。在hp端粒周围的复制会产生通过复制端粒（rTel）连接连接在一起的环状二聚体子dna。为了使子dna在子细胞之间分裂，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，分离线性子DNA，在线性DNA末端再生新的hp端粒。******我的实验室研究ResT的特性。在本提案中，我们将检查/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合成的延续中。在最近的实验室进展中，我们发现ResT具有意想不到的生化特性，允许它分离DNA链并重新退火它们。我们还发现ResT与一种促进相同DNA拷贝之间遗传信息交换的蛋白质有关。我们将该蛋白命名为RarA （rest相关重组酶A）。这些活动与许多生物体中参与修复DNA损伤和修复DNA损伤部位的DNA复制复合体的蛋白质非常相似。******我们假设这些修复酶的相似性表明，hp端粒看起来像DNA损伤的细胞，并且需要ResT/RarA提供的功能来完成伯氏疏螺旋体线性DNA的复制。我们将通过观察试管和实验室培养的疏螺旋体中蛋白质的特性来验证我们的假设。我们将在试管中比较ResT和RarA与已知修复酶的特性，以确定其与已知修复功能的相似程度。然后我们将干扰细胞中这些蛋白质的功能，看看这是否会干扰细胞复制DNA的能力。如果这些蛋白质具有类似DNA修复的功能对细胞存活很重要，那么它们将构成伯氏疏螺旋体中第一个确定的修复功能的例子。以前人们认为疏螺旋体缺乏这些功能。对酵母和哺乳动物细胞线性DNA复制过程中类似基本问题的研究，极大地促进了我们对细胞转化和衰老等基本生物学问题的理解。

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.