Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Biochemistry, Cellular and Molecular Biology Graduate Program, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Nucleic Acids Res. 2021 Apr 19;49(7):3967-3980. doi: 10.1093/nar/gkab206.
In budding yeast, Rif1 negatively regulates telomere length, but the mechanism of this regulation has remained elusive. Previous work identified several functional domains of Rif1, but none of these has been shown to mediate telomere length. To define Rif1 domains responsible for telomere regulation, we localized truncations of Rif1 to a single specific telomere and measured telomere length of that telomere compared to bulk telomeres. We found that a domain in the N-terminus containing HEAT repeats, Rif1177-996, was sufficient for length regulation when tethered to the telomere. Charged residues in this region were previously proposed to mediate DNA binding. We found that mutation of these residues disrupted telomere length regulation even when Rif1 was tethered to the telomere. Mutation of other conserved residues in this region, which were not predicted to interact with DNA, also disrupted telomere length maintenance, while mutation of conserved residues distal to this region did not. Our data suggest that conserved amino acids in the region from 436 to 577 play a functional role in telomere length regulation, which is separate from their proposed DNA binding function. We propose that the Rif1 HEAT repeats region represents a protein-protein binding interface that mediates telomere length regulation.
在芽殖酵母中， Rif1 负调控端粒长度，但这种调控的机制仍不清楚。以前的工作确定了 Rif1 的几个功能域，但没有一个被证明介导端粒长度。为了确定 Rif1 调节端粒的结构域，我们将 Rif1 的截断定位到单个特定的端粒上，并测量该端粒与大量端粒的端粒长度。我们发现，包含 HEAT 重复序列的 Rif1177-996 N 端结构域在与端粒连接时足以进行长度调节。该区域的带电残基以前被提议介导 DNA 结合。我们发现，即使 Rif1 与端粒连接，这些残基的突变也会破坏端粒长度的调节。该区域其他保守残基的突变，这些残基不被预测与 DNA 相互作用，也破坏了端粒长度的维持，而该区域以外的保守残基的突变则没有。我们的数据表明，该区域 436 到 577 位的保守氨基酸在端粒长度调节中发挥功能作用，这与它们提出的 DNA 结合功能是分开的。我们提出 Rif1 的 HEAT 重复区代表一个蛋白质-蛋白质结合界面，介导端粒长度的调节。
