Site-specific ADP-ribosylation of histone H2B in response to DNA double strand breaks.
Site-specific ADP-ribosylation of histone H2B in response to DNA double strand breaks.
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DOI：
10.1038/srep43750
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发表时间：
2017-03-02
影响因子：
4.6
通讯作者：
Lakin ND
中科院分区：
文献类型：
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作者：
Rakhimova A;Ura S;Hsu DW;Wang HY;Pears CJ;Lakin ND
ADP-核糖基转移酶（ART）用ADP-核糖的单个单元或聚合物修饰蛋白质以调节DNA修复。然而，这些酶的底物不明确。例如，虽然组蛋白被ART修饰，但这些蛋白质上的位点在DNA损伤后ADP-核糖基化，以及催化这些事件的ART是未知的。这在一定程度上是由于缺乏含有ART的真核模型，以及可以被操纵以评估体内ADP-核糖基化事件的组蛋白基因。在这里，我们利用模型Dictyosteoblasts，以确定在体内的位点特异性组蛋白ADP-核糖基化事件，并定义的ART介导这些修改。Dictyosteoblasts组蛋白在体内被ART Adprt 1a和Adprt 2修饰以响应DNA双链断裂（DSB）。Adprt 1a是一种在体外修饰H2 BE 18的单ART，尽管该位点的破坏允许H2 BE 19处的ADP-核糖基化。虽然H2 BE 18和H2 BE 19 ADP核糖基化之间的冗余在体内DSB后也是明显的，但通过产生在h2 b基因座中的E18/E19处具有突变的菌株，我们证明这些是Adprt 1a/Adprt 2修饰的主要位点。这确定了DNA损伤诱导的组蛋白单ADP核糖基化位点的特定ART在体内，提供了一个独特的平台，以评估组蛋白ADP核糖基化如何调节DNA修复。
ADP-ribosyltransferases (ARTs) modify proteins with single units or polymers of ADP-ribose to regulate DNA repair. However, the substrates for these enzymes are ill-defined. For example, although histones are modified by ARTs, the sites on these proteins ADP-ribosylated following DNA damage and the ARTs that catalyse these events are unknown. This, in part, is due to the lack of a eukaryotic model that contains ARTs, in addition to histone genes that can be manipulated to assess ADP-ribosylation events in vivo. Here we exploit the model Dictyostelium to identify site-specific histone ADP-ribosylation events in vivo and define the ARTs that mediate these modifications. Dictyostelium histones are modified in response to DNA double strand breaks (DSBs) in vivo by the ARTs Adprt1a and Adprt2. Adprt1a is a mono-ART that modifies H2BE18 in vitro, although disruption of this site allows ADP-ribosylation at H2BE19. Although redundancy between H2BE18 and H2BE19 ADP-ribosylation is also apparent following DSBs in vivo, by generating a strain with mutations at E18/E19 in the h2b locus we demonstrate these are the principal sites modified by Adprt1a/Adprt2. This identifies DNA damage induced histone mono-ADP-ribosylation sites by specific ARTs in vivo, providing a unique platform to assess how histone ADP-ribosylation regulates DNA repair.