Weitao Tao, Budd Martin, Campbell Judith L
Braun Laboratories 147-75, California Institute of Technology, Pasadena, CA 91125, USA.
Mutat Res. 2003 Nov 27;532(1-2):157-72. doi: 10.1016/j.mrfmmm.2003.08.015.
We and others have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We showed previously that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the hypomorphic dna2-2 helicase mutant. Deletion of FOB1 suppresses the elevated pausing and DSB formation. Our current work shows that mutation inactivating Sgs1, the yeast RecQ helicase ortholog, also causes accumulation of stalled replication forks and DSBs at the rDNA RFB. Either deletion of FOB1, which suppresses fork blocking and certain types of rDNA recombination, or an increase in SIR2 gene dosage, which suppresses rDNA recombination, reduces the number of forks persisting at the RFB. Although dna2-2 sgs1Delta double mutants are conditionally lethal, they do not show enhanced rDNA defects compared to sgs1Delta alone. However, surprisingly, the dna2-2 sgs1Delta lethality is suppressed by deletion of FOB1. On the other hand, the dna2-2 sgs1Delta lethality is only partially suppressed by deletion of rad51Delta. We propose that the replication-associated defects that we document in the rDNA are characteristic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.
我们和其他研究人员曾提出，停滞的复制叉处理错误会导致酿酒酵母中的重组增加和染色体不稳定。现在，我们利用核糖体DNA位点（它是DNA复制各个阶段的良好模型）来检验这一假设。我们之前表明，核糖体DNA复制叉屏障（RFB）处的DNA复制暂停伴随着RFB附近双链断裂的出现。在低表达的dna2 - 2解旋酶突变体中，暂停和断裂都有所增加。FOB1的缺失抑制了增加的暂停和双链断裂的形成。我们目前的研究表明，使酵母RecQ解旋酶直系同源物Sgs1失活的突变也会导致rDNA RFB处停滞的复制叉和双链断裂的积累。要么缺失抑制叉阻塞和某些类型rDNA重组的FOB1，要么增加抑制rDNA重组的SIR2基因剂量，都会减少在RFB处持续存在的叉的数量。虽然dna2 - 2 sgs1Δ双突变体是条件致死的，但与单独的sgs1Δ相比，它们并未表现出增强的rDNA缺陷。然而，令人惊讶的是，FOB1的缺失抑制了dna2 - 2 sgs1Δ的致死性。另一方面，rad51Δ的缺失仅部分抑制了dna2 - 2 sgs1Δ的致死性。我们提出，我们在rDNA中记录的与复制相关的缺陷是整个基因组中随机发生的类似事件的特征，或者是在复制叉移动缓慢或停滞的其他区域（如端粒、着丝粒或复制缓慢区）发生的类似事件的特征。
