Mechanisms of Genome Integrity

 描述(申请人提供)：我们的染色体不断地受到各种侮辱的轰炸，导致损坏，必须修复。细胞必然会进化出检测和修复DNA断链的机制，从而防止重要遗传信息的丢失。双链DNA断裂(DSB)是一种会导致特别灾难性后果的损伤。如果不加以纠正，DSB可能会导致严重的染色体重排，这是所有形式癌症的标志。令人惊讶的是，DNA复制是DSB的主要来源。同源重组(HR)是细胞修复双链断裂的一条高度保守的途径，对于防止和修复DNA复制过程中的损伤是必要的。当DSB发生时，DNA末端被处理以产生3‘单链DNA(SsDNA)突出物。然后，单链DNA末端与基因组其他地方的同源序列配对，并使用同源DNA作为复制模板来替换缺失的DNA序列。最后，复制的中间体被分解，重新获得断裂DNA的连续性。虽然看起来很简单，但HR需要一系列复杂的蛋白质的协调行动，这些蛋白质负责感知损伤，招募必要的因素，以及处理和修复受损的DNA。扰乱人力资源的后果是毁灭性的。例如，RAD51重组酶的突变对小鼠是胚胎致命的，而人类RAD51的突变与乳腺癌有关。此外，BRCA2基因缺陷至少占所有乳腺癌的5%，也是卵巢癌的遗传易感性。BRCA2被认为有助于调节HR，而这一调节的缺失可能是该基因与遗传性癌症有关的原因。为了充分理解这些结果的机制基础，重大的新发现将是必要的。我们的整体研究计划侧重于了解(I)蛋白质如何感知和响应受损的DNA，(Ii)DNA损伤是如何修复的，(Iii)DNA复制如何导致损伤，以及(Iv)复制和重组是如何联系在一起的。为了帮助解决这些问题，我们开发了独特的技术，使我们能够使用光学显微镜直接显示数百个单独的分子，这使我们能够在单分子水平上实时监控DNA修复和DNA复制的空间和时间进程。使用这种方法，我们试图定义我们的细胞用来复制和修复DNA的基本机制，长期目标是了解这些过程中的错误如何导致染色体重排。

 DESCRIPTION (provided by applicant): Our chromosomes are continually bombarded with a variety of insults, resulting in damage that must be repaired. By necessity, cells have evolved mechanisms to detect and repair broken strands of DNA, thereby preventing loss of important genetic information. Double-stranded DNA breaks (DSBs) are a type of damage that led to particularly disastrous outcomes. If not corrected, DSBs can lead to gross chromosomal rearrangements, which are the hallmark of all forms of cancer. Surprisingly, DNA replication is the primary source of DSBs. Homologous recombination (HR) is a highly conserved pathway that cells can use to repair DSBs, and HR is necessary to prevent and repair the damage that arises during DNA replication. When a DSB occurs, the DNA ends are processed to generate 3' single-strand DNA (ssDNA) overhangs. The ssDNA ends then pair with homologous sequence elsewhere in the genome, and the missing DNA sequence is replaced using the homologous DNA as a template for replication. Finally, the replicated intermediate is resolved, regenerating the continuity of the broken DNA. While seemingly simple, HR requires the coordinated action of a complex repertoire of proteins, which are responsible for sensing damage, recruiting essential factors, and processing and repairing the damaged DNA. The consequences of disrupting HR are devastating. For example, mutations in the Rad51 recombinase are embryonic lethal in mice, and mutations in human Rad51 are linked to breast cancers. In addition, defects in BRCA2 account for at least 5% of all breast cancers and also confer a genetic predisposition to ovarian cancer. BRCA2 is thought to help regulate HR, and loss of this regulation may be the reason why this gene is linked to hereditary cancers. Major new discoveries will be necessary to fully understand the mechanistic basis for these outcomes. Our overall research program is focused on understanding how (i) proteins sense and respond to damaged DNA, (ii) how DNA damage is repaired, (iii) how DNA replication can lead to damage, and (iv) how replication and recombination are linked. To help address these problems we have developed unique technologies that allow us to directly visualize hundreds of individual molecules using optical microscopy, which enables us to monitor the spatial and temporal progression of DNA repair and DNA replication in real-time at the single-molecule level. Using this approach we seek to define the fundamental mechanisms that our cells use to replicate and repair DNA, with the long-term goal of understanding how errors during these processes can lead to chromosomal rearrangements.