Multiple DNA Repair Pathways in Recombinational Process

DNA修复和重组是细胞对环境和内源性应激反应的基本过程。了解参与DNA修复和重组的基因的功能对于理解诱变的分子基础和基因重排、有丝分裂重组和致癌的起源至关重要。在项目4中，我们将通过实验确定哺乳动物DNA修复基因的一个子集，核苷酸切除修复（NER），双链断裂修复（DSBR）和错配修复（MMR）途径的组成部分之间的一些关系，这些基因已知在修复和重组中发挥作用。我们推测，许多由这些基因编码的蛋白质在哺乳动物细胞中的DNA链间交联（ICLs）和其他类型的复杂DNA损伤的修复过程中的多个步骤中发挥作用。项目4的具体重点是这些基因在重组修复中的功能。在酵母中，RAD（辐射敏感）基因参与多种DNA修复途径，包括NER和DSBR，也构成重组修复途径。对酵母的研究为哺乳动物RAD同系物在哺乳动物细胞中的修复和重组功能提供了线索，但在DNA修复和重组机制方面，低等和高等真核生物之间也存在重要差异;此外，对哺乳动物细胞修复复杂病变（如ICLs）的了解甚少。哺乳动物细胞中修复和重组的复杂性增加反映在许多RAD基因的旁系同源物以及真正的直系同源物的数量上。这些组件形成不同的 复合物在修复和重组中起作用，并且与酵母相比，可能赋予哺乳动物DNA修复过程显着和微妙的差异。在项目4中，我们将构建具有特异性缺陷基因（即，ERCC 1、XPF、RAD 52、MSH 3、XRCC 3和RAD 51 C）;这些细胞系将含有染色体内APRT直接重复重组底物，并且相应的等基因细胞系将含有适合作为重组靶标的APRT等位基因。这些专门的细胞系将用于研究和比较ICL和DSB的重组修复，重点是RAD 51依赖性和非依赖性重组修复途径， 用于通过上位性实验研究遗传相互作用。项目4将产生结果，解决哺乳动物细胞中复杂病变的修复涉及在多个步骤中起作用的多个修复途径的组成部分的总体假设，包括NER，DSBR和MMR途径的元素。

DNA repair and recombination are fundamental processes underlying cellular responses to environmental and endogenous stresses. Understanding the functions of genes involved in DNA repair and recombination is critical to understanding the molecular basis of mutagenesis and the origins of gene rearrangements, mitotic recombination and carcinogenesis. In Project 4, we will experimentally define some of the relationships among a subset of mammalian DNA repair genes, components of the nucleotide excision repair (NER), double-strand break repair (DSBR), and mismatch repair (MMR) pathways, which are known to function in both repair and recombination. We hypothesize that many of the proteins encoded by these genes function at multiple steps in repair processing of DNA interstrand crosslinks (ICLs) and other types of complex DNA damage in mammalian cells. The specific focus of Project 4 is the function of these genes in recombinational repair. In yeast, the RAD (radiation-sensitive) genes participate in a variety of DNA repair pathways, including NER and DSBR, and also constitute recombinational repair pathways. Studies in yeast have provided clues to how mammalian RAD homologs function in repair and recombination in mammalian cells, but there are also important differences between lower and higher eukarotes in DNA repair and recombination mechanisms; also, repair of complex lesions such as ICLs by mammalian cells is poorly understood. The increased complexity of both repair and recombination in mammalian cells is reflected in the number of paralogs, as well as true orthologs, of many RAD genes. These components form different complexes functioning in repair and recombination, and likely impart both significant and subtle differences to mammalian DNA repair processes compared to yeast. In Project 4, we will construct DNA repairproficient and -deficient CHO cell lines with specifically defective genes (i.e., ERCC1, XPF, RAD52, MSH3, XRCC3 and RAD51C); these cell lines will contain intrachromosomal APRT direct-repeat recombination substrates, and corresponding isogenic cell lines will contain APRT alleles suitable as recombination targets. These specialized cell lines will be used to investigate and compare recombinational repair of ICLs and DSBs, focusing on RAD51-dependent and -independent recombinational repair pathways, and will also be used to investigate genetic interactions through epistasis experiments. Project 4 will generate results addressing the overall hypothesis that repair of complex lesions in mammalian cells involves components of multiple repair pathways acting at multiple steps, including elements of NER, DSBR, and MMR pathways.