Break-induced replication and genome rearrangements

描述（由申请人提供）： 摘要染色体双链断裂（DSB）是在正常细胞代谢过程中自发发生的细胞毒性损伤，或通过用DNA损伤剂处理细胞而发生。如果未修复或修复不当，DSB可能导致诱变事件，如染色体丢失、缺失、复制或易位，这些事件可能导致致癌作用。通过同源重组（HR）修复DSB依赖于同源双链体的存在以模板修复断裂的染色体，并且通常被认为是无错误的机制。然而，如果重组序列不相同，HR可导致局部杂合性丢失（洛），如果修复与染色体同源物之间的交叉相关，则可导致广泛的洛。此外，如果在异位位点处的重复序列被用作序列供体并且重组与交换相关，则可能发生易位。当DSB的两端与供体双链体序列共享同源性时，HR通过两端机制进行，导致主要为非交换产物。然而，如果两端的协调没有保持，或者只有一端的断裂是可用的，例如在一个非常短的端粒，修复可以发生 断裂诱导复制（break induced replication，BIR）在这种情况下，随后的链侵入复制可以延伸超过100 kb以到达染色体的末端。如果入侵发生在一个分散的重复序列上，这可能导致广泛的洛缺失或非相互易位。在这个建议中，我们将继续机制的研究，以了解如何洛和染色体重排发生的BIR或通过决议的重组中间体使用酵母模型系统。具体目标是：（1）监测染色体DSB的BIR修复的新系统将用于确定DNA合成的机制并测试ssDNA中间体的不稳定性降低BIR效率的想法。此外，我们计划使用iPOND方法通过与新生DNA链的关联来识别参与BIR的新因子。(2)我们将建立一种新的检测方法来检测人工或天然重复序列之间的模板转换，并确定调节这一过程的基因。(3)我们将确定结构选择性核酸酶对DSB诱导的和自发的有丝分裂交叉的错误调节的后果，并跟踪有丝分裂过程中未解决的重组中间体的命运。也将确定错配修复，Rad 1-Rad 10和Mph 1解旋酶在控制交叉中的作用。

DESCRIPTION (provided by applicant): SUMMARY Chromosomal double strand breaks (DSBs) are cytotoxic lesions that occur spontaneously during normal cell metabolism or by treatment of cells with DNA-damaging agents. If unrepaired or repaired inappropriately, DSBs can lead to mutagenic events, such as chromosome loss, deletions, duplications or translocations, events that can lead to carcinogenesis. The repair of DSBs by homologous recombination (HR) relies on the presence of a homologous duplex to template repair of the broken chromosome and is generally considered to be an error-free mechanism. However, HR can lead to a local loss of heterozygosity (LOH) if the recombining sequences are not identical, and to extensive LOH if repair is associated with a crossover between chromosome homologs. Furthermore, if a repeated sequence at an ectopic site is utilized as the sequence donor and recombination is associated with crossing over, translocations can occur. When both ends of the DSB share homology with the donor duplex sequence, HR proceeds by a two-ended mechanism resulting in primarily non-crossover products. However, if coordination of the two ends is not maintained or only one end of the break is available, such as at a critically short telomere, repair can occur by break-induced replication (BIR). In this case, following strand invasion replication can extend for more than 100 kb to reach the end of the chromosome. This can cause extensive LOH or non-reciprocal translocation if invasion occurs at a dispersed repeated sequence. In this proposal we will continue mechanistic studies to understand how LOH and chromosome rearrangements occur by BIR or by resolution of recombination intermediates using the yeast model system. The specific aims are: (1) A new system to monitor BIR repair of a chromosomal DSB will be used to determine the mechanism of DNA synthesis and test the idea that destabilization of the ssDNA intermediate decreases BIR efficiency. In addition, we plan to use the iPOND method to identify new factors involved in BIR by association with nascent DNA strands. (2) We will establish a new assay to detect template switching between artificial or natural repeats and identify genes that regulate this process. (3) We will determine the consequences of mis-regulation of structure-selective nucleases on DSB induced and spontaneous mitotic crossovers, and follow the fate of unresolved recombination intermediates during mitosis. The roles of mismatch repair, Rad1-Rad10 and the Mph1 helicase in controlling crossovers will also be determined.