Thongsroy Jirapan, Matangkasombut Oranart, Thongnak Araya, Rattanatanyong Prakasit, Jirawatnotai Siwanon, Mutirangura Apiwat
Inter-Department Program of BioMedical Sciences, Faculty of Graduate School, Chulalongkorn University, Bangkok, Thailand.
PLoS One. 2013 Aug 19;8(8):e72706. doi: 10.1371/journal.pone.0072706. eCollection 2013.
Without exposure to any DNA-damaging agents, non-dividing eukaryotic cells carry endogenous DNA double-strand breaks (EDSBs), or Replication-Independent (RIND)-EDSBs. In human cells, RIND-EDSBs are enriched in the methylated heterochromatic areas of the genome and are repaired by an ATM-dependent non-homologous end-joining pathway (NHEJ). Here, we showed that Saccharomyces cerevisiae similarly possess RIND-EDSBs. Various levels of EDSBs were detected during different phases of the cell cycle, including G0. Using a collection of mutant yeast strains, we investigated various DNA metabolic and DNA repair pathways that might be involved in the maintenance of RIND-EDSB levels. We found that the RIND-EDSB levels increased significantly in yeast strains lacking proteins involved in NHEJ DNA repair and in suppression of heterochromatin formation. RIND-EDSB levels were also upregulated when genes encoding histone deacetylase, endonucleases, topoisomerase, and DNA repair regulators were deleted. In contrast, RIND-EDSB levels were downregulated in the mutants that lack chromatin-condensing proteins, such as the high-mobility group box proteins, and Sir2. Likewise, RIND-EDSB levels were also decreased in human cells lacking HMGB1. Therefore, we conclude that the genomic levels of RIND-EDSBs are evolutionally conserved, dynamically regulated, and may be influenced by genome topology, chromatin structure, and the efficiency of DNA repair systems.
在不接触任何DNA损伤剂的情况下，非分裂真核细胞携带内源性DNA双链断裂（EDSBs），即复制非依赖性（RIND）-EDSBs。在人类细胞中，RIND-EDSBs在基因组的甲基化异染色质区域富集，并通过依赖ATM的非同源末端连接途径（NHEJ）进行修复。在此，我们表明酿酒酵母同样具有RIND-EDSBs。在细胞周期的不同阶段，包括G0期，都检测到了不同水平的EDSBs。我们使用一系列突变酵母菌株，研究了可能参与维持RIND-EDSB水平的各种DNA代谢和DNA修复途径。我们发现，在缺乏参与NHEJ DNA修复和抑制异染色质形成的蛋白质的酵母菌株中，RIND-EDSB水平显著增加。当编码组蛋白脱乙酰酶、核酸内切酶、拓扑异构酶和DNA修复调节因子的基因被删除时，RIND-EDSB水平也会上调。相反，在缺乏染色质凝聚蛋白（如高迁移率族框蛋白和Sir2）的突变体中，RIND-EDSB水平下调。同样，在缺乏HMGB1的人类细胞中，RIND-EDSB水平也降低。因此，我们得出结论，RIND-EDSBs的基因组水平在进化上是保守的，受到动态调节，并且可能受到基因组拓扑结构、染色质结构和DNA修复系统效率的影响。
