Recovery of Replication Following UV-induced DNA damage

在存在DNA损伤的情况下，不准确的复制常常导致细胞重排和突变。然而，复制阻断病变处理和修复的细胞机制在很大程度上仍不清楚。紫外线诱导的DNA损伤严重抑制染色体复制。在大肠杆菌中，紫外线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，一些额外的基因可能参与复制恢复，但它们尚未在体内直接检查。该项目将确定染色体复制在被紫外线诱导的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确定病变停止复制分叉的结构特性。该项目的这一部分将通过在蔗糖梯度中从紫外线突变体中分离辐照后DNA来完成，以定量在复制停止发生之前，DNA复制机制可以绕过（或跳过）紫外线诱导的病变的数量。辐照后的DNA合成将使用链特异性32p标记探针进行分析，以确定在哪个模板链上发生复制阻断。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，并识别在恢复过程中出现在阻断复制叉的结构中间体。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，并描述它们在哪个步骤起作用。这将通过分离在CsCl梯度中复制恢复期间合成的DNA并对其进行量化来完成。这些候选基因在复制恢复中发挥的作用将进一步通过检查在紫外线照射后阻断复制分叉处的新生DNA降解来表征。通过理解被DNA损伤阻断的忠实复制是如何恢复的，研究人员希望确定在DNA损伤存在的情况下导致非法恢复、基因组重排和致命性的条件和事件。因为DNA的变化会影响生物学的各个方面，这些见解可能会深刻地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.