Recovery of Replication Following UV-induced DNA damage

存在DNA损伤时的不准确复制通常会导致细胞重排和突变。 然而，细胞机制的复制阻断病变的处理和修复仍然在很大程度上没有特点。 紫外线诱导的DNA损伤严重抑制染色体复制。 在大肠杆菌中，UV照射后复制的恢复依赖于核苷酸切除修复蛋白和recF途径的一些蛋白。 据推测，几个额外的基因可能参与复制恢复，但他们还没有在体内直接检查。 该项目将确定当染色体复制被紫外线诱导的DNA损伤阻断时恢复的细胞机制。 研究者将在整个复制恢复过程中识别病变停滞复制叉的结构特性。 该项目的这一部分将通过在蔗糖梯度中从uvr突变体中分离辐照后的DNA来完成，以定量在复制停滞发生之前可以被DNA复制机器绕过（或跳过）的UV诱导的损伤的数量。 然后使用链特异性32P标记的探针分析辐照后的DNA合成，以确定发生模板链复制阻断。此外，研究者将使用二维琼脂糖凝胶电泳来表征整个恢复过程中复制叉的结构完整性，并鉴定恢复过程中阻断复制叉处出现的结构中间体。 他将进一步确定哪些候选基因直接参与DNA损伤阻断复制叉的复制恢复，并描述它们在哪一步起作用。 这将通过分离在CsCl梯度中复制恢复期间合成的DNA并对其进行定量来完成。这些候选基因在复制恢复中发挥的作用将通过检查UV照射后阻断复制叉处的新生DNA降解来进一步表征。 通过了解当被DNA损伤阻断时忠实复制如何恢复，研究人员希望确定可能导致DNA损伤存在下的非法恢复、基因组重排和致死性的条件和事件。 由于DNA的变化影响生物学的各个方面，这些见解可能会深刻影响人类与自然世界的互动。

Inaccurate replication in the presence of DNA damage is often responsible for cellular rearrangements and mutagenesis. Nevertheless, the cellular mechanisms by which replication-blocking lesions are processed and repaired remain largely uncharacterized. UV-induced DNA damage severely inhibits chromosomal replication. In Escherichia coli, the recovery of replication following UV irradiation is dependent on nucleotide excision repair proteins and some proteins of the recF pathway. It is speculated that several additional genes may be involved in replication recovery but they have not been examined directly in vivo. This project will determine the cellular mechanism by which chromosomal replication recovers when it is blocked by UV-induced DNA damage. The investigator will identify the structural properties of lesion-arrested replication forks throughout replication recovery. This portion of the project will be done by isolating the post-irradiation DNA from uvr mutants in sucrose gradients to quantitate the number of UV-induced lesions that can be bypassed (or skipped) by DNA replication machinery before replication arrest occurs. Post-irradiation DNA synthesis will then be analyzed using strand-specific 32P-labeled probes to determine on which template strand replication blockage occurs. In addition, the investigator will use two-dimensional agarose gel electrophoresis to characterize the structural integrity of the replication fork throughout the recovery process and identify the structural intermediates that arise at blocked replication forks during recovery. He will further identify which candidate genes are directly involved in the resumption of replication at DNA damage-blocked replication forks and characterize at which step they act. This will be done by isolating DNA synthesized during the period of replication recovery in CsCl gradients and quantifying it. The role that these candidate genes play in replication recovery will further be characterized by examining the nascent DNA degradation at blocked replication forks following UV irradiation. Through an understanding of how faithful replication resumes when it is blocked by DNA damage, the investigator hopes to identify the conditions and events which can lead to illegitimate recovery, genomic rearrangements, and lethality in the presence of DNA damage. Because changes in DNA affect all aspects of biology, these insights could profoundly affect human interactions with the natural world.