Biochemical and Structural Analysis of CHD-class chromatin remodelers

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。

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the nine CHD enzymes, CHD2, CHD3 and CHD4 all have well described roles in DNA damage response, and our laboratory has unpublished evidence for both CHD5 and CHD6 playing major roles in the oxidative DNA damage response.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.