Biochemical and Structural Analysis of CHD-class chromatin remodelers

基因组DNA与组蛋白一起包装，形成一个复杂的梯度，从松弛的常染色质到高度浓缩的异染色质--这种分布是我们基因组相对于原核祖先大幅增加的大小和复杂性的基础。结构复杂的染色质抑制DNA加工酶，包括解决DNA损伤所需的那些因子。如果不能准确及时地修复DNA损伤，可能会导致基因序列改变和基因组不稳定。真核生物有复杂的手段来访问和操纵染色质内结合的DNA，特别是在细胞创伤的时候。ATP依赖的染色质重塑酶可以调节核小体之间的连接DNA间距的长度，以调节DNA可及性，并且对于所有真核生物中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赋予核小体重新间隔、去除或交换活性的位于中心的ATP酶/解旋酶结构域。在9种CHD酶中，CHD 2，CHD 3和CHD 4都在DNA损伤反应中有很好的描述作用，我们的实验室有未发表的证据表明CHD 5和CHD 6在氧化DNA损伤反应中起主要作用。关于人类CHD酶的结构和生物化学信息很少，仅可获得人类CHD 2、4和5的酶活性数据，并且仅解析了CHD 421 -23片段的结构数据。没有关于CHD 6的活性或结构的任何信息，或者在受控条件下每种CHD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一个主要盲点。

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the nine CHD enzymes, CHD2, CHD3 and CHD4 all have well described roles in DNA damage response, and our laboratory has unpublished evidence for both CHD5 and CHD6 playing major roles in the oxidative DNA damage response.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