REGULATION OF THE BREAST CELL DNA SYNTHESOME

乳腺细胞 DNA 合成体的调节

• 批准号: 6124559
• 负责人: ROBERT J HICKEY
• 金额: $ 19.75万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-18 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6124559
• 关键词: ADP ribosylation; DNA directed DNA polymerase; DNA replication; SDS polyacrylamide gel electrophoresis; adenine phosphoribosyltransferase; breast neoplasms; cell line; chemical kinetics; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; gel mobility shift assay; mammary gland; molecular weight; nucleic acid biosynthesis

DESCRIPTION: The genetic damage which accompanies the development and progression of breast cancer has been linked to defects in the DNA synthetic and DNA repair processes of these cells; and these defects correlate with changes in the physical structure and enzymatic activity of several proteins used to carry-out DNA synthesis and repair. The investigator's data indicate that, as compared to normal breast cells, breast cancer cells exhibit a 3-4-fold higher DNA synthetic rate, a 6-8-fold decrease in replication fidelity, and have specific structural alterations in several of the proteins used to carry-out DNA replication. His goal, therefore, is to establish a clear link between the differences in the DNA synthetic activity and replication fidelity of normal and malignant breast cells and the structural changes specific replication essential proteins undergo during transformation. To accomplish this, he will initially focus on defining the kinetic, biophysical, and detailed structural properties of the DNA polymerases and poly(ADP-ribose) polymerase (PARP) by determining: 1) differences in the kinetic properties of these enzymes between the normal and malignant breast cells looking specifically at the Km and Vmax for substrate utilization, substrate specificity, and the proof-reading activity of the intrinsic 3-5' exonuclease of DNA polymerases delta and epsilon; as well as the fidelity with which all of the DNA polymerases carry-out DNA synthesis; 2) differences in the biophysical characteristics of normal and malignant breast cell DNA polymerases alpha, delta, epsilon and PARP looking specifically at the molecular weight, Stokes radii, sedimentation coefficient, isoelectric point, and 2D-PAGE profiles of these enzymes; and 3) differences in the 2D-PAGE tryptic peptide maps of the normal and malignant breast cell polymerases looking specifically at the role of phosphorylation, poly(ADP-ribosylation), and primary amino acid sequence in mediating these differences.

描述：伴随发育和发育而来的遗传损害 乳腺癌的进展与DNA合成缺陷有关 以及这些细胞的DNA修复过程；这些缺陷与 几种蛋白质的物理结构和酶活性的变化 用于进行DNA合成和修复。调查员的数据 表明，与正常乳腺细胞相比，乳腺癌细胞 表现出3-4倍的DNA合成速度，6-8倍的下降 复制保真度，并在几个 用来进行DNA复制的蛋白质。因此，他的目标是 在DNA合成活动的不同之间建立明显的联系 以及正常和恶性乳腺细胞的复制保真度 特定复制必需蛋白在体内经历结构变化 转型。为了实现这一点，他首先将重点放在定义 DNA的动力学、生物物理和详细结构特性 聚合酶和聚(ADP-核糖)聚合酶(PARP)测定：1) 这些酶的动力学性质在正常情况下的差异 恶性乳腺细胞专门观察Km和Vmax 底物利用、底物专一性和校对活性 DNA聚合酶Delta和epsilon的固有3-5‘外切酶；AS 以及所有DNA聚合酶携带DNA的保真度 2)正常和正常的生物物理特性的差异。 恶性乳腺细胞DNA聚合酶α、β、epsilon和PARP的研究 特别是在相对分子质量、斯托克斯半径、沉淀物 这些酶的系数、等电点和2D-PAGE图谱；以及 3)正常人和正常人胰酶2D-PAGE图谱的差异 恶性乳腺细胞聚合酶在乳腺癌中的作用 磷酸化、多(ADP-核糖化)和初级氨基酸序列 调停这些分歧。