ABSTRACT

Cellular genomes undergo various structural changes that include cis tethering (the tethering together of two loci within a single DNA molecule), which promotes chromosome condensation and transcriptional activation, and trans tethering (the tethering together of two DNA molecules), which promotes sister chromatid cohesion and DNA repair. The protein complex termed cohesin promotes both cis and trans forms of DNA tethering, but the extent to which these cohesin functions occur in temporally or spatially defined contexts remains largely unknown. Prior studies indicate that DNA polymerase sliding clamp PCNA recruits cohesin acetyltransferase Eco1, suggesting that sister chromatid cohesion is established in the context of the DNA replication fork. In support of this model, elevated levels of PCNA rescue the temperature growth and cohesion defects exhibited by eco1 mutant cells. Here, we test whether Eco1-dependent chromatin condensation is also promoted in the context of this DNA replication fork component. Our results reveal that overexpressed PCNA does not promote DNA condensation in eco1 mutant cells, even though Smc3 acetylation levels are increased. We further provide evidence that replication fork-associated E3 ligase impacts on Eco1 are more complex that previously described. In combination, the data suggests that Eco1 acetylates Smc3 and thus promotes sister chromatid cohesion in context of the DNA replication fork, whereas a distinct cohesin population participates in chromatin condensation outside the context of the DNA replication fork.

 抽象的

细胞基因组经历各种结构变化，包括顺式束缚（单个 DNA 分子内两个基因座束缚在一起），促进染色体浓缩和转录激活，以及反式束缚（两个 DNA 分子束缚在一起），促进姐妹染色单体凝聚力和DNA修复。称为粘连蛋白的蛋白质复合物促进顺式和反式DNA 束缚，但这些粘连蛋白功能在时间或空间定义的背景下发生的程度仍然很大程度上未知。先前的研究表明DNA聚合酶滑夹PCNA招募粘连蛋白乙酰转移酶Eco1，这表明姐妹染色单体的粘连是在DNA复制叉的背景下建立的。为了支持这一模型，升高的 PCNA 水平可以挽救eco1突变细胞表现出的温度生长和内聚缺陷。在这里，我们测试了 Eco1 依赖性染色质浓缩是否也在 DNA 复制叉成分的背景下得到促进。我们的结果表明，即使 Smc3 乙酰化水平增加，过表达的 PCNA 也不会促进eco1突变细胞中的 DNA 浓缩。我们进一步提供证据表明复制叉相关的 E3 连接酶对 Eco1 的影响比之前描述的更为复杂。综合起来，数据表明 Eco1 乙酰化 Smc3，从而促进 DNA 复制叉背景下的姐妹染色单体凝聚，而独特的凝聚素群体则参与 DNA 复制叉背景外的染色质凝聚。