A conserved MCM single-stranded DNA binding element is essential for replication initiation.
A conserved MCM single-stranded DNA binding element is essential for replication initiation.
复制标题
保守的MCM单链DNA结合元件对于复制启动至关重要。
DOI：
10.7554/elife.01993
复制
发表时间：
2014-04-01
期刊：
影响因子：
7.7
通讯作者：
Enemark EJ
中科院分区：
文献类型：
--
作者：
Froelich CA;Kang S;Epling LB;Bell SP;Enemark EJ
环状MCM解旋酶对DNA复制的所有阶段都是必不可少的。该复合体在复制起始处以不活跃的双六聚体的形式包围着双链DNA。解旋酶的激活将该物种转化为两个围绕单链DNA(SsDNA)的活性单一六聚体。这些事件期间MCM DNA相互作用的分子细节尚不清楚。我们测定了与单链DNA结合的狂热杆菌MCM N-末端结构域六聚体的晶体结构，并定义了一个保守的MCM-单链DNA结合基序(MSSB)。有趣的是，ssDNA以确定的极性结合了垂直于中央通道的MCM环内部。在真核生物中，MSSB在几个Mcm2-7亚基中是保守的，而酿酒酵母Mcm2-7中的MSSB突变组合是不可行的。突变的Mcm2-7复合体可以组装并被招募到复制起点，但在解旋酶的装载和激活方面存在缺陷。我们的发现确定了一个重要的MCM-ssDNA相互作用，并表明它在解旋酶激活过程中起作用，选择转位的链。DOI：http://dx.doi.org/10.7554/eLife.01993.001当dna第一次被识别为双螺旋时，很明显，这种结构可以很容易地解释dna如何复制。每条链由碱基组成--用字母‘A’、‘C’、‘G’和‘T’表示--两条链通过一对碱基之间的键连接在一起，每条碱基一个。此外，“A”总是与“T”配对，而“C”总是与“G”配对。因此，如果两条链被分离，每条链都可以作为模板来指导新的互补链的合成，从而创建原始双链分子的两个副本。这个复制过程的第一步包括一个由六种蛋白质组成的环形复合体，称为MCM解旋酶，将两条链分开。为了准备DNA复制，两个MCM解旋酶环包裹在双链DNA周围。然后，在解旋酶被激活后，DNA碱基对之间的键断裂，两个环分离，一环环绕每条DNA链。然而，解旋酶和DNA在这些事件中相互作用的细节还不完全清楚。现在，Froelich，Kang等人。已经解决了MCM解旋酶环的三维结构--取自最初在深海喷口发现的微生物--本身以及与一小段单链DNA结合时也是如此。当DNA与解旋酶环结合时，解旋酶环变得更加椭圆形。此外，DNA不是直接穿过环，而是包裹环内的一部分。环内侧的特定氨基酸--蛋白质的组成成分--与单链DNA相互作用，这些氨基酸也存在于许多其他生物体的MCM蛋白质中。此外，将这些氨基酸交换为不同的氨基酸显著降低了环与单链DNA的结合能力，但其与双链DNA的结合能力仅受到轻微影响。在酵母细胞的环状复合体中设计类似的变化是致命的，突变复合体更不能被加载到DNA上，或者被激活并分离准备复制的两条链。这些对解旋酶如何加载到双链DNA上，并选择一条DNA链进行环绕的见解，提高了我们对DNA复制是如何启动的理解：这是一个对生物至关重要的过程。DOI：http://dx.doi.org/10.7554/eLife.01993.002
The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001 When DNA was first recognised to be a double helix, it was clear that this structure could easily explain how DNA could be replicated. Each strand was made of bases—represented by the letters ‘A’, ‘C’, ‘G’ and ‘T’—and the two strands were held together by bonds between pairs of bases, one from each strand. Moreover, ‘A’ always paired with ‘T’, and ‘C’ always paired with ‘G’. Therefore, if the two strands were separated, each could be used as a template to guide the synthesis of a new complementary strand and thus create two copies of the original double-stranded molecule. One of the first steps in this replication process involves a ring-shaped complex of six proteins, called an MCM helicase, separating the two strands. To prepare for DNA replication, two MCM helicase rings wrap around the double-stranded DNA. Then, after the helicase has been activated, the bonds between the DNA base pairs break, and the two rings separate with one ring encircling each DNA strand. However, the details of the interactions between the helicase and the DNA during these events are not fully understood. Now Froelich, Kang et al. have solved the three-dimensional structure of an MCM helicase ring—taken from a microbe originally found at deep ocean vents—on its own and also when bound to a short piece of single-stranded DNA. The helicase ring becomes more oval when the DNA binds to it. Moreover, rather than passing straight through the ring, the DNA wraps part of the way around the inside of the ring. Specific amino acids—the building blocks of proteins—on the inside of the ring interact with the single-stranded DNA, and these amino acids are also found in MCM proteins in many other organisms. Furthermore, swapping these amino acids for different amino acids significantly reduced the ability of the ring to bind to single-stranded DNA, but its ability to bind to double-stranded DNA was only slightly affected. Engineering similar changes into the ring complexes of yeast cells was lethal, and the mutant complexes were less able to be loaded onto the DNA, or to be activated and separate the two strands ready for replication. These insights into how helicases are loaded onto double-stranded DNA, and select one DNA strand to encircle, have improved our understanding of how DNA replication is initiated: a process that is vital for living things. DOI: http://dx.doi.org/10.7554/eLife.01993.002