Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA.
Proc Natl Acad Sci U S A. 2010 Dec 14;107(50):21605-10. doi: 10.1073/pnas.1001938107. Epub 2010 Nov 23.
Genomic rearrangements are common, occur by largely unknown mechanisms, and can lead to human diseases. We previously demonstrated that some genome rearrangements occur in budding yeast through the fusion of two DNA sequences that contain limited sequence homology, lie in inverted orientation, and are within 5 kb of one another. This inverted repeat fusion reaction forms dicentric chromosomes, which are well-known intermediates to additional rearrangements. We have previously provided evidence indicating that an error of stalled or disrupted DNA replication forks can cause inverted repeat fusion. Here we analyze how checkpoint protein regulatory pathways known to stabilize stalled forks affect this form of instability. We find that two checkpoint pathways suppress inverted repeat fusion, and that their activities are distinguishable by their interactions with exonuclease 1 (Exo1). The checkpoint kinase Rad53 (Chk2) and recombination protein complex MRX(MRN) inhibit Exo1 in one pathway, whereas in a second pathway the ATR-like kinases Mec1 and Tel1, adaptor protein Rad9, and effector kinases Chk1 and Dun1 act independently of Exo1 to prevent inverted repeat fusion. We provide a model that indicates how in Rad53 or MRX mutants, an inappropriately active Exo1 may facilitate faulty template switching between nearby inverted repeats to form dicentric chromosomes. We further investigate the role of Rad53, using hypomorphic alleles of Rad53 and null mutations in Rad9 and Mrc1, and provide evidence that only local, as opposed to global, activity of Rad53 is sufficient to prevent inverted repeat fusion.
基因组重排很常见，其发生机制尚不清楚，可能导致人类疾病。我们之前证明，在酿酒酵母中，一些基因组重排是通过两个含有有限序列同源性、反向排列且彼此相距 5kb 以内的 DNA 序列融合而发生的。这种反向重复融合反应形成了双中心染色体，这是进一步重排的众所周知的中间体。我们之前提供的证据表明，停滞或中断的 DNA 复制叉的错误可能导致反向重复融合。在这里，我们分析了已知能稳定停滞叉的检查点蛋白调节途径如何影响这种不稳定性。我们发现，两条检查点途径抑制反向重复融合，并且它们的活性可以通过与核酸外切酶 1（Exo1）的相互作用来区分。检查点激酶 Rad53（Chk2）和重组蛋白复合物 MRX（MRN）在一条途径中抑制 Exo1，而在另一条途径中，ATR 样激酶 Mec1 和 Tel1、衔接蛋白 Rad9 和效应激酶 Chk1 和 Dun1 独立于 Exo1 发挥作用，以防止反向重复融合。我们提供了一个模型，表明在 Rad53 或 MRX 突变体中，不适当活跃的 Exo1 可能会促进附近反向重复之间错误的模板转换，从而形成双中心染色体。我们进一步研究了 Rad53 的作用，使用 Rad53 的弱等位基因和 Rad9 和 Mrc1 的 null 突变，并提供证据表明，只有局部而非全局的 Rad53 活性足以防止反向重复融合。
