Genetic and molecular mechanisms of replication of araC damaged DNA

 描述(申请人提供)：阿糖胞苷(β-D-arabinofanosyl cytosine，ARAC)用于治疗急性髓系白血病已有40多年。AraCTP与dCTP竞争掺入DNA；因此，Arac的化疗作用源于其抑制DNA复制的能力。复制聚合酶(Pols)虽然可以在新合成的DNA的3‘端插入araCTP，但它们在从其延伸时受到抑制。然而，人类细胞含有一些跨病变合成(TLS)DNA POL，原则上可以通过延长Arac末端的DNA合成和通过并入模板链的Arac损伤进行复制来克服Arac对DNA复制的抑制作用。为了了解TLS POLS促进Arac损伤DNA复制的相对意义和机制，我们将进行遗传学、细胞学、生化和结构研究相结合的研究。在目标1中，我们将(A)分析TLS Pol在介导Arac在人类细胞中的复制中的作用，并确定它们是以无错误还是以突变的方式起作用；(B)检测Arac损伤的DNA复制所需的TLS Pol的缺失对Arac处理的人细胞中复制叉进展的影响；以及(C)检测TLS Pol的缺失对Arac处理的人细胞存活的影响。在目标2中，我们将进行稳态动力学分析，以(A)确定TLS Pol对从3‘端的Arac延伸的催化效率；(B)测定TLS Pol对与Arac相对的核苷酸(NT)掺入的熟练程度和保真度；以及(C)分析TLS Pol从插入的NT相对Arac延伸的熟练程度；此外，我们将进行稳定前的动力学研究，以获得更好的 对TLS Pol从Arac延伸和插入NTS与Arac相反的机制的理解。在目标3中，我们将确定TLS POLS的三元复杂晶体结构：(A)在模板链中与Arac相对的位置插入NTS；(B)当Arac与引物末端配对时，通过插入正确或不正确的NT来发挥TLS延伸步骤的功能；以及(C)从Arac末端的DNA中延伸DNA合成所需的结构。综上所述，这些研究将使人们更深入地了解不同的TLS POL在促进Arac损伤DNA复制方面所起的作用，以及TLS POL如何设法将Arac适应于其活性部位及其动力学作用机制。除了关于TLS Pol在Arac受损DNA复制中的作用的新的机制信息外，这些研究还可能提出提高Arac化疗在复发是主要问题的癌症治疗中的有效性的方法。

 DESCRIPTION (provided by applicant): Cytarabine (β-D-arabinofuranosyl cytosine, araC) has been used for the treatment of acute myelogenous leukemia (AML) for over 40 years. AraCTP competes with dCTP for incorporation into DNA; thus, the chemotherapeutic action of araC derives from its ability to inhibit DNA replication. Although the replicative polymerases (Pols) ca insert araCTP at the 3' terminus of newly synthesized DNA, they are inhibited at extending from it. However, human cells harbor a number of translesion synthesis (TLS) DNA Pols that can, in principle, overcome the inhibitory effects of araC on DNA replication by both extending DNA synthesis from araC-terminated 3' ends and by replicating through the araC lesion that becomes incorporated into the template strand. To understand the relative significance and mechanisms of TLS Pols in promoting replication of araC-damaged DNA, we will carry out a combination of genetic, cellular, biochemical, and structural studies. In Aim 1, we will (a) analyze the roles of TLS Pols in mediating replication through araC in human cells and determine whether they act in an error-free or mutagenic manner; (b) examine the effects of depletions of TLS Pols required for the replication of araC-damaged DNA on the progression of the replication fork in araC treated human cells; and (c) examine the effects of depletions of TLS Pols on the survival of human cells treated with araC. In Aim 2, we will carry out steady- state kinetic analyses to (a) determine the catalytic efficiencies of TLS Pols for extending from araC at the 3' primer terminus; (b) determine the proficiency and fidelity of TLS Pols for nucleotide (nt) incorporation opposite araC; and (c) analyze the proficiency of TLS Pols for extending from the nt inserted opposite araC; in addition, (d) we will carry out pre-steady state kinetic studies to gain a better understanding of the mechanisms of TLS Pols in extending from araC and in inserting nts opposite araC. In Aim 3, we will determine ternary complex crystal structures of TLS Pols that (a) insert nts opposite araC in the templating strand; (b) that function in the extension step of TLS by inserting the correct or incorrect nt when araC is paired to the primer terminus; and (c) that are required for the extension of DNA synthesis from araC terminated DNA. Altogether, these studies will provide a deeper understanding of the roles that different TLS Pols play in promoting the replication of araC damaged DNA, and how the TLS Pols manage to accommodate araC into their active sites and their kinetic mechanisms of action. In addition to novel mechanistic information on the roles of TLS Pols in the replication of araC damaged DNA, these studies may posit ways to increase the effectiveness of araC chemotherapy for the treatment of a cancer where relapse is a major problem.