MUTATIONAL SIGNATURE(S) OF HUMAN GENOMIC REARRANGEMENT MECHANISMS

项目摘要/摘要 拷贝数变异(CNV)，即基因组某一片段的太多或太少拷贝，是导致许多 重要的人类医学问题。我们曾预测，基于我们的MMBIR模型， 许多CNV是通过异常修复断裂的复制叉来实现的，因此会有一条单向的 从新冠病毒交界处延伸出来的相当长的超突变。我们设计了一系列 分析17号染色体上CNV周围的700万个碱基对DNA序列的技术。 通过这些技术，我们成功地确定了26的精确结构和与之相关的突变 CNV。我们还分析了父母双方的基因组。我们找到了我们想要的：有大片的 与CNV连锁的一个方向的超突变，从CNV延伸到100万个碱基对。这 证实我们的假设机制至少要为这些CNV事件的一半负责。对于另一半来说 ，我们将从更大的父母样本中获得数据，直到我们可以确定 这些是由不同的机制产生的，还是由同一机制分布的尾巴造成的。 由于我们分析的非常详细的解决方案，我们能够看到 产生超突变区域。我们找到了两种我们可以暂时破译的机制，可能还有一个 第三个原因我们还没有找到。在CNV附近，低加工能力的聚合酶使多个 正在复制的模板上的模板切换和滑动。在更远的地方，我们看到了 作用于单链DNA的过程产生了独特特征的簇状突变。第三个签名 可能与中断复制派生主复制时预期的失配修复减少有关。 额外的数据将使签名更加清晰，并使我们能够确定原因。下一步是 将这些发现推广到基因组的其余部分。我们将通过CNV的全基因组测序来完成这项工作，网址为 其他网站。在第三个项目中，我们将使用新的测序技术来破译复发的CNV 这是通过重复序列之间的交叉而产生的。这一直是以前的一个棘手的问题 技术，因为细胞中存在大量的序列副本。我们希望找到准确的 交叉和基因转换区域的位置，指示控制它们将落在哪里的规则，以及 确定任何新突变的范围和特征(从而确定原因)。这些目标加在一起将 扩展对DNA修复事件的理解，这些事件会导致基因组疾病，并可能 建议控制或避免这种情况发生的方法。

PROJECT SUMMARY/ABSTRACT Copy number variation (CNV), too many or too few copies of a segment of the genome, underlies many important human medical issues. We had predicted that, based on our MMBIR model for the generation of much CNV by aberrant repair of broken replication forks, that there would be a unidirectional tract of hypermutation of considerable length extending from the junction of the CNV. We devised a series of techniques to analyze seven million base-pair tracts of DNA sequence surrounding CNVs on chromosome 17. With these techniques we were successful in determining the precise structure of, and mutations linked to, 26 CNVs. We also analyzed both parents' genomes. We discovered what we sought: there are tracts of hypermutation in one direction linked to the CNV extending for up to one million base-pairs from the CNV. This confirms that our postulated mechanism is responsible for at least half of these CNV events. For the other half that do not show hypermutation, we will obtain data from a larger sample of parents until we can say whether these arose by a different mechanism or whether it is the tail of the distribution of the same mechanism. Because of the very highly detailed resolution of our analyses, we are able to see into the mechanisms that generate the hypermutation tracts. We find two mechanisms that we can provisionally decipher and possibly a third whose cause we have not yet found. Near the CNV, a low processivity polymerase makes multiple template switches and slips on the template that is being replicated. Further away, we see evidence of processes acting on single-stranded DNA giving clustered mutations of a unique signature. The third signature might relate to the diminished mismatch repair that is expected when broken replication forks prime replication. The additional data will make the signatures clearer and allow us to determine the causes. The next step is to generalize the findings to the rest of the genome. We will do this by whole genome sequencing of CNVs at other sites. In a third project we are going to use new sequencing technology to decipher the recurrent CNVs that arise by crossing-over between repeated sequences. This has been an intractable problem with previous technology because of the numerous copies of the sequence present in the cell. We expect to find the precise positions of crossovers and gene conversion tracts, indicating the rules that govern where they will fall, and determine the extent and signature (and therefore the cause) of any new mutations. Together these Aims will extend understanding of DNA repair events gone wrong that lead to genomic disorders, and potentially suggest ways to control or avoid this happening.