Error Prone vs Error Free DNA Replication in Human Cells

人类细胞中容易出错的 DNA 复制与无错误的 DNA 复制

• 批准号: 6522675
• 负责人: VERONICA M MAHER
• 金额: $ 34.66万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6522675
• 关键词: DNA damage; DNA directed DNA polymerase; DNA repair; DNA replication; complementary DNA; enzyme activity; fibroblasts; gene conversion; genetic recombination; human tissue; hypoxanthine phosphoribosyltransferase; nucleic acid sequence; polymerase chain reaction; tissue /cell culture

The overall goals of the proposed studies are: to examine in human cells the mechanisms involved in "translesion synthesis"(TLS), i.e., the process by which human cells insert nucleotides opposite lesions in the DNA template that block replication by the major replication complex; to examine the mechanisms involved in "damage avoidance", a broad term that refers to various error-free processes by which human cells complete replication without using the damaged DNA as a template; and to determine whether there is a reciprocal relationship between these two alternative pathways. 1.) We will test the hypothesis that when replication by the normal replication complex is blocked by lesions, TLS, using the damaged DNA as a template, involves the products of several specialized human polymerases and the chance that a specific polymerase will introduce "spontaneous" or mutagen-induced mutations during TLS depends upon: a) the nature of the lesion; b) whether it is located in the template strand for leading or lagging strand synthesis; c) the surrounding sequence; and d) the availability of the other competing specialized polymerases in the cell; 2.) We will test the hypothesis that when DNA replication is blocked, human fibroblasts obtain the necessary genetic information from an undamaged homologous copy of the DNA, e.g., the newly- replicated daughter-strand of the corresponding DNA, i.e., the sister chromatid or the corresponding allele (recombination, gene conversion); 3.) We will test the hypothesis that if such "damage avoidance" pathways for obtaining the genetic information from homologous copies of DNA are blocked, use of mutagenic TLS will increase correspondingly, i.e., the relationship is reciprocal; 4.) We will determine a) the nature of the "damage-avoidance" mechanism(s) used to complete lesion-blocked DNA replication b) the relative frequency at which cells use those pathways rather than TLS pathways, and c) the effect of lack of particular gene products on the (reciprocal) relationship between the pathways, using substrates designed to reveal the mechanisms used, from the nature of the products.

拟议的研究的总体目标是：在人类细胞中检查“跨性别合成”（TLS）所涉及的机制，即人类细胞插入核苷酸在DNA模板中插入核苷酸相反的核苷酸，从而通过主要复制复合物阻断复制的核苷酸；为了检查“避免损害”所涉及的机制，这是指人类细胞在不使用受损DNA作为模板的情况下完成复制的各种过程的广义术语；并确定这两个替代途径之间是否存在相互关系。 1.）我们将检验以下假设：当通过损害的DNA用作模板的病变（TLS）封闭正常复制复合物的复制时，涉及几种专业人类聚合酶的产物，并且特定聚合酶会引入“自发性”或Mutagen诱导的TLS依赖于：A）的可能性。 b）它是否位于模板链中，用于领先还是滞后链合成； c）周围序列； d）细胞中其他竞争专业聚合酶的可用性； 2.）我们将检验以下假设：当DNA复制被阻断时，人成纤维细胞从DNA的未损坏的同源副本中获得必要的遗传信息，例如，新近复制的相应DNA的新复制的女子链，即姐妹染色质酸或相应的等位基因（重组，基因转化）； 3.）我们将检验以下假设：如果从DNA的同源副本中获取遗传信息的“避免损害”途径被阻断，则使用诱变TLS将相应地增加，即这种关系是相互倒数的； 4.）我们将确定a）用于完成病变的DNA复制的“避免损伤”机制的性质b）细胞使用这些途径而不是TLS途径的相对频率，以及c）缺乏特定基因产物对使用该途径的途径之间使用的（相互互动）关系的影响，该途径使用设计用于使用该机制的基础，该途径揭示了使用的机制。