SIR2 Suppresses Replication Gaps and Genome Instability in Yeast by Balancing Replication Between Repetitive and Unique Sequences. Saccharomyces cerevisiae

Replication gaps that persist into mitosis likely represent important threats to genome stability, but experimental identification of these gaps has proved challenging. We have developed a technique that allows us to explore the dynamics by which genome replication is completed prior to mitosis. Using this approach, we demonstrate that excessive allocation of replication resources to origins within repetitive regions, induced by SIR2 deletion, leads to persistent replication gaps and genome instability. Conversely, the weakening of replication origins in repetitive regions suppresses these gaps. Given known age- and cancer-associated changes in chromatin accessibility at repetitive sequences, we suggest that replication gaps resulting from misallocation of replication resources underlie age- and disease-associated genome instability. Overall design: flow sort yeast according to DNA content into G1, S, early G2 and late G2, isolate DNA, and subject DNA to Illumina single end sequencing

持续至有丝分裂（mitosis）的复制间隙（replication gaps）可能对基因组稳定性（genome stability）构成重大威胁，但实验层面识别此类间隙却颇具挑战。我们开发了一项技术，可用于探究基因组复制（genome replication）在有丝分裂前完成的动态机制。依托该技术，我们证实：由SIR2基因缺失诱导的、向重复区域内复制起始位点（replication origins）过度分配复制资源的行为，会引发持续性复制间隙与基因组不稳定。反之，削弱重复区域内的复制起始位点则可抑制此类间隙的产生。鉴于目前已知的、与衰老和癌症相关的重复序列区域染色质可及性（chromatin accessibility）改变，我们提出：因复制资源分配失调所产生的复制间隙，是衰老与疾病相关基因组不稳定的核心诱因。实验整体设计：依据DNA含量通过流式分选将酵母（yeast）分为G1、S、早G2与晚G2期细胞，提取DNA后对其进行Illumina单端测序（single end sequencing）。