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Author:
Date:
2018-10-05
[Abstract] The Small Ubiquitin-related Modifier (SUMO) is a protein that is post-translationally added to and reversibly removed from other proteins in eukaryotic cells. SUMO and enzymes of the SUMO pathway are well conserved from yeast to humans and SUMO modification regulates a variety of essential cellular processes including transcription, chromatin remodeling, DNA damage repair, and cell cycle progression. One of the challenges in studying SUMO modification in vivo is the relatively low steady-state level of a SUMO-modified protein due in part to the activity of SUMO deconjugating enzymes ...
[摘要] 小泛素相关修饰物（SUMO）是一种蛋白质，其翻译后添加到真核细胞中并可逆地从其他蛋白质中去除。 SUMO和SUMO途径的酶从酵母到人类都很保守，SUMO修饰调节了多种基本细胞过程，包括转录，染色质重塑，DNA损伤修复和细胞周期进程。 研究SUMO修饰体内的挑战之一是SUMO修饰蛋白的相对低的稳态水平，部分原因是SUMO去缀合酶（SUMO Isopeptidases或SENPs）的活性。 幸运的是，使用重组SUMO酶可以在体外研究SUMO修饰。 在这里，我们描述了一种灵敏的方法，用于检测目标人类蛋白质的SUMO修饰，使用来自兔网织红细胞和放射性标记的氨基酸的体外转录和翻译系统。
【背景】与其他泛素蛋白家族修饰一样，SUMO修饰通过ATP依赖性酶促级联发生，涉及E1激活酶（人类中的Aos1 / Uba2异二聚体），E2结合酶（Ubc9）和许多E3连接之一的连续活性。酶（Gareau和Lima，2010）。具有SUMO缀合共有位点的蛋白质ΨKxE（Ψ是疏水残基，其后是赖氨酸，任何氨基酸和谷氨酸），可以通过哺乳动物中表达的一种或几种SUMO旁系同源物（包括SUMO1，SUMO2）进行有效修饰。或SUMO3（统称为SUMO2 / 3，因为它们的序列同源性为97％）（Gareau和Lima，2010; Flotho和Melchior，2013）。 ...
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Author:
Date:
2017-09-20
[Abstract] The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other proteins. We have previously established the mechanisms by which the polymerases ε and δ are targeted to their ‘correct’ strands, as well as quality control mechanisms that evict polymerases when they associate with an ‘incorrect’ strand. Here, we ...
[摘要] 真核生物复制品是重复DNA的多蛋白复合物。 复制品被雕刻成连续的前导链合成与不连续的滞后链合成，主要通过DNA聚合酶ε和δ以及解旋酶，聚合酶α-引发酶，DNA滑动夹，夹带载体和许多其它蛋白质进行。 我们以前已经建立了聚合酶ε和δ靶向其“正确”链的机制，以及在与“不正确”链相关联时驱赶聚合酶的质量控制机制。 在这里，我们提供了使用纯蛋白质在体外差异测定前导和滞后链复制的实用指南。
Using pure proteins from Saccharomyces cerevisiae, our lab was the first to reconstitute a functional eukaryotic DNA replisome, a ~2 MDa complex that includes the 11-subunit CMG helicase (complex of Cdc45, Mcm2-7, GINS heterotetramer), the 4-subunit DNA polymerase (Pol) ε, the 4-subunit Pol α-primase, the PCNA (Proliferating Cell Nuclear Antigen) clamp homotrimer ring shaped processivity factor that ...
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