The structures of the SNM1A and SNM1B/Apollo nuclease domains reveal a potential basis for their distinct DNA processing activities.
The structures of the SNM1A and SNM1B/Apollo nuclease domains reveal a potential basis for their distinct DNA processing activities.
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DOI：
10.1093/nar/gkv1256
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发表时间：
2015-12-15
影响因子：
14.9
通讯作者：
Gileadi O
中科院分区：
文献类型：
--
作者：
Allerston CK;Lee SY;Newman JA;Schofield CJ;McHugh PJ;Gileadi O
人类SNM1a和SNM1B/Apollo蛋白是一个真核核酸酶家族的成员，该家族含有一个与原核生物金属β内酰胺酶折叠相关的基序。SNM1A是复制依赖和转录偶联的链间交联修复过程中的关键核酸外切酶，而SNM1B/Apollo是维持端粒悬垂所必需的。在此，我们报道了SNM1A和SNM1B的晶体结构。虽然这两种蛋白都含有一个典型的MBL-β-CASP结构域，但SNM1a的活性部位周围有一个正电荷区域，这在SNM1B中是不存在的，这解释了SNM1a具有更强的表观处理能力。这两种蛋白质的结构也显示出一个假定的、广泛的DNA结合槽。对该凹槽进行了广泛的突变，再加上详细的生化分析，确定了不影响SNM1A催化活性但极大地降低了其加工性的残基。此外，我们还发现了这种沟槽在有效消化通过DNA链间交联键，促进SNM1A催化的关键DNA修复反应中的关键作用。综上所述，这种沟槽的结构和尺寸，再加上周围高正电荷的区域，解释了SNM1A适应和有效消化高度扭曲的DNA底物的非凡能力，例如那些包含DNA损伤的底物。
The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-β-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-β-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions.