Biophysics of Macromolecular Complexes

我们对天然染色质片段的生物物理性质和结构感兴趣。鸡叶酸受体和β-珠蛋白基因座是此类结构研究的理想区域，因为(I)该区域具有浓缩的和转录活性的染色质区域，以及(Ii)该系统在基因调控方面已被广泛研究，使我们能够将整个染色质结构与转录联系起来。浓缩的染色质区域跨越16kbp的DNA，两侧是发育调节的叶酸受体和β-珠蛋白基因，可以通过限制酶HpaII从细胞核中释放出来。我们以前已经将生物物理方法与实时荧光聚合酶链式反应和Southern blotting相结合，分析了这个16kbp的浓缩染色质的流体力学性质，表明它是一个延伸的杆状结构。这提供了对散布在不同基因组中的异染色质结构的洞察。 我们利用我们开发的方法进一步研究了天然染色质片段。使用红系前体细胞系，我们已经分析了转录稳定的β-珠蛋白基因位点的16KBP区域，并表明该染色质片段似乎也是一个延伸的杆状。然而，与浓缩的染色质片段不同，这个染色质区域的蛋白质与核酸的比例较低，这表明RNA在这个复合体的稳定中发挥了作用。对10日龄鸡红细胞染色质的类似研究正在进行中，其中β-珠蛋白基因座在转录上是活跃的。所研究的染色质片段的持续长度(灵活性)提供了额外的信息，我们目前正在开发生物化学方法来确定这种珠蛋白基因染色质片段的灵活性。 大分子组装。 与分子生物学实验室和其他实验室的成员合作，利用流体力学方法对蛋白质和蛋白质-核酸组合体的形状、化学计量和相互作用的亲和力进行了表征。这些方法补充了生化和结构研究，并提供了有关生物学机制的重要信息。最近与魏阳博士一起对保守的MutL蛋白进行的研究就是一个很好的例子。我们证明了MutL的C-末端结构域是一个二聚体。此外，我们还表明，连接N-末端和C-末端的富含Pro的连接体基本上是刚性的，导致杆状蛋白质的端到端距离大于10 nm。结合其他数据，这些研究为大肠杆菌中的DNA错配修复提供了一个工作模型。

We are interested in the biophysical properties and structure of native chromatin fragments. The chicken folate receptor and beta-globin gene loci are ideal for such structural studies in that (i) the region possesses both condensed and transcriptionally active chromatin regions, and (ii) the system has been extensively studied in terms of gene regulation, allowing us to relate the overall chromatin structure to transcription. The condensed chromatin region, spanning 16 kbp of DNA flanked by the developmentally regulated folate receptor and beta-globin genes, can be released from the cell nucleus with the restriction enzyme HpaII. We have previously combined biophysical methods with real-time PCR and Southern blotting to analyze the hydrodynamic properties of this 16 kbp condensed chromatin and showed that it is an extended rod. This provides insights into the structure of heterochromatin, found interspersed within various genomes. We have furthered our studies of native chromatin fragments utilizing the methods we developed. Using an erythroid precursor cell line, we have analyzed a 16 kbp region of the transcriptionally poised beta-globin gene locus and showed that this chromatin fragment also appears to be an extended rod. However, unlike the condensed chromatin fragment, this chromatin region has a lower protein to nucleic acid ratio suggesting a role for RNA in the stabilization of this complex. Similar studies on 10-day old chicken erythrocyte chromatin, in which the beta-globin locus is transcriptionally active, are in progress. The persistence length (flexibility) of the chromatin fragments studied provides additional information and we are currently developing biochemical methods to determine the flexibility of such globin gene chromatin fragments. Macromolecular assemblies. In collaboration with members of the Laboratory of Molecular Biology, and other laboratories, protein and protein-nucleic acid assemblies have been characterized in terms of their shape, stoichiometry and affinity of interaction using hydrodynamic methods. These methods complement biochemical and structural investigations and provide important information on the biological mechanism. A case in point is provided by the recent studies of the conserved MutL protein carried out with Dr. Wei Yang. We showed that the C-terminal domain of MutL is a dimer. Furthermore, we showed that the proline-rich linker connecting the N- and C-termini is essentially rigid resulting in a greater than 10 nm end-to-end distance for the rod-shaped protein. Together with other data, these studies provide a working model for DNA mismatch repair in E. coli.