Biochemical and Structural Analysis of CHD-class chromatin remodelers

我的研究项目旨在了解参与DNA双链断裂反应的CHD类染色质重塑酶的生化活性、结构生物学和分子编排。基因组DNA与组蛋白蛋白包装在一起，形成一个复杂的梯度，从相对松弛的常染色质到高度浓缩的异染色质，这一分布导致了相对于我们的原核祖先，我们基因组的大小和复杂性都大大增加了。结构复杂的染色质对DNA处理酶有抑制作用，包括那些解决DNA损伤所需的因子。如果不能准确和及时地修复DNA损伤，可能会导致基因序列改变和基因组不稳定。真核生物有复杂的方法来访问和操纵结合在染色质中的DNA，特别是在细胞损伤的时候。依赖于ATP的染色质重塑酶可以调节核小体之间连接DNA间距的长度，从而调节DNA的可及性，对于所有真核生物的基因组稳定性是必不可少的。染色体结构域-解旋酶-DNA结合(CHD)染色质重塑酶具有两个染色质结构域和一个中心定位的ATPase/解旋酶结构域，赋予核小体重新排列、移除或交换活性。在9种CHD酶中，CHD2、CHD3和CHD4都在DNA损伤反应中发挥了很好的作用，我们的实验室也有未发表的证据表明CHD5和CHD6在DNA氧化损伤反应中发挥重要作用。有关人类CHD酶的结构和生化信息很少，酶活性数据仅适用于人类CHD2、4和5，结构数据仅适用于CHD421-23片段。没有关于CHD6的活性或结构的任何信息，或者在受控条件下每个CHD酶的活性如何相互比较，这是我们对这个重要的酶家族知识的主要盲点。 我们认为，鉴于五种不同的CHD酶是DNA损伤反应的已知参与者，现在是时候对这一染色质重构体家族如何在有基因组损伤的细胞中协同发挥作用并相互影响进行全面分析了。我们将：(1)纯化人CHD2、CHD3、CHD4、CHD5和CHD6，并鉴定它们的酶活性和底物的相对选择性。利用小角X射线散射(SAXS)和X射线结晶学，我们还将(2)开发有关CHD酶的溶液中和静态结构信息。我们将(3)利用微辐射和活细胞成像，确定每个CHD酶相对于彼此的招募和扩散到DNA损伤和扩散的精确编排。最后，我们将(4)监测CHD联合消融对细胞命运的影响。这些科学努力将是第一次对参与DNA双链断裂反应的每一种人类冠心病酶进行全面的比较分析。

My RESEARCH PROGRAM aims to understand the biochemical activity, structural biology and molecular choreography of CHD-class chromatin remodeling enzymes involved in the DNA double strand break response. Genomic DNA is packaged with histone proteins to form a complex gradient of comparably relaxed euchromatin to highly condensed heterochromatin a distribution underlying the hugely increased size and complexity of our genome relative to our prokaryotic ancestors. Structurally complex chromatin is inhibitory to DNA processing enzymes, including those factors required to resolve DNA damage. Failure to repair DNA damage in an accurate and timely manner can lead to gene sequence alterations and genome instability. Eukaryotes have complex means of accessing and manipulating DNA bound within chromatin, particularly in times of cell trauma. ATP-dependent chromatin remodeling enzymes can adjust the length of linker DNA spacing between nucleosomes to regulate DNA accessibility, and are essential for genome stability in all eukaryotes. Chromodomain-Helicase-DNA binding (CHD) chromatin remodeling enzymes have double chromodomains and a centrally-positioned ATPase/helicase domain that confers nucleosome re-spacing, removal or exchange activity. Among the nine CHD enzymes, CHD2, CHD3 and CHD4 all have well described roles in DNA damage response, and our laboratory has unpublished evidence for both CHD5 and CHD6 playing major roles in the oxidative DNA damage response. Structural and biochemical information on human CHD enzymes is sparse, with enzymatic activity data only available for human CHD2, 4 and 5 and structural data only resolved for fragments of CHD421-23. No information whatsoever on the activity or structure of CHD6 is available, or how the activity of each CHD enzyme compares with one another under controlled conditions a major blind spot in our knowledge of this important enzyme family. We suggest that the time is now right, given that five distinct CHD enzymes are known participants in the DNA damage response, to undertake a comprehensive analysis of how this family of chromatin remodelers function in unison and impact one another in cells with genomic damage. We will: (1) purify human CHD2, 3, 4, 5 and 6 and characterize their enzymatic activity and substrate preferences relative to one another. Using small angle x-ray scattering (SAXS) and x-ray crystallography, we will also (2) develop in-solution and static structural information on CHD enzymes. We will (3) ascertain the precise choreography of recruitment and dispersal of each CHD enzyme to and from DNA damage, relative to one another, using micro-irradiation and live-cell imaging. Finally, we will (4) monitor the cell fate impact of combinatorial CHD ablation. These scientific endeavors will represent the first comprehensive comparative analysis of each human CHD enzyme involved in the DNA double strand break response.