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、结构生物学和分子舞蹈。基因组DNA被组蛋白包裹，形成了一个复杂的梯度，从相对宽松的常染色质到高度浓缩的异染色质，这种分布是我们的基因组相对于我们的原核祖先的大小和复杂性大大增加的基础。结构复杂的染色质抑制DNA加工酶，包括解决DNA损伤所需的那些因素。不能准确及时地修复DNA损伤会导致基因序列改变和基因组不稳定。真核生物具有访问和操纵染色质内结合的DNA的复杂方法，特别是在细胞创伤的时候。atp依赖性染色质重塑酶可以调节核小体之间连接体DNA间距的长度来调节DNA的可及性，对所有真核生物的基因组稳定性至关重要。染色体结构域-解旋酶- dna结合（CHD）染色质重塑酶具有双染色体结构域和一个位于中心位置的atp酶/解旋酶结构域，该结构域赋予核小体重新间隔、移除或交换活性。在9种冠心病酶中，CHD2、CHD3和CHD4都在DNA损伤反应中有很好的作用，我们实验室有未发表的证据表明CHD5和CHD6在DNA氧化损伤反应中起主要作用。人类冠心病酶的结构和生化信息很少，酶活性数据仅针对人类CHD2、4和5，结构数据仅针对CHD421-23片段。没有任何关于CHD6活性或结构的信息，或者在受控条件下每种冠心病酶的活性如何相互比较-这是我们对这一重要酶家族知识的主要盲点。******我们认为现在是时候了，鉴于已知有五种不同的冠心病酶参与DNA损伤反应，对这一染色质重塑家族如何在基因组损伤细胞中一致发挥作用并相互影响进行全面分析。我们将：(1)纯化人CHD2、3、4、5和6，并表征它们的酶活性和相对于它们的底物偏好。利用小角x射线散射（SAXS）和x射线晶体学，我们还将(2)开发冠心病酶的溶液内和静态结构信息。我们将(3)利用微辐射和活细胞成像技术，确定每一种冠心病酶相对于DNA损伤的招募和分散的精确编排。最后，我们将(4)监测冠心病联合消融对细胞命运的影响。这些科学努力将首次对参与DNA双链断裂反应的每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the nine CHD enzymes, CHD2, CHD3 and CHD4 all have well described roles in DNA damage response, and our laboratory has unpublished evidence for both CHD5 and CHD6 playing major roles in the oxidative DNA damage response.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.**