The 11th C2H2 zinc finger and an adjacent C-terminal arm are responsible for TZAP recognition of telomeric DNA
The 11th C2H2 zinc finger and an adjacent C-terminal arm are responsible for TZAP recognition of telomeric DNA
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第 11 个 C2H2 锌指和相邻的 C 端臂负责端粒 DNA 的 TZAP 识别
DOI：
10.1038/cr.2017.141
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发表时间：
2018
期刊：
影响因子：
44.1
通讯作者：
Li Fudong
中科院分区：
文献类型：
--
作者：
Zhao Yaqing;Zhang Guang;He Chao;Mei Yide;Shi Yunyu;Li Fudong
端粒长度的内稳态决定着细胞的增殖潜能，对正常的细胞功能至关重要。除了在细胞分裂过程中众所周知的端粒缩短和端粒酶激活或端粒延长（ALT）机制的延长外，端粒长度也受到被称为端粒修剪[1]的调节快速缺失事件的影响。这一修剪过程涉及到被称为t环的端粒结构的切除，这需要同源重组蛋白XRCC3和NBS1[2,3]。最近的研究表明，端粒修剪和延长之间的平衡决定了种系细胞和干细胞的端粒长度，这表明端粒修剪应该受到严格的控制[4,5]。然而，端粒修剪的调控在很大程度上仍然是未知的。最近，两组研究人员建立了人类TZAP （ZBTB48，重命名为端粒锌指相关蛋白）作为直接结合双链端粒TTAGGG序列并刺激端粒修剪的因子[6-9]。TZAP由一个n端BTB/POZ结构域和其c端11个相邻的c2h2型锌指（Znf1-11）组成（图1A）。Li等人[6]将TZAP的TTAGGG结合区定位为Znf9-11，而进一步的研究将其细化为Znf11[7]。据我们所知，这是第一次报道C2H2指直接结合端粒DNA，尽管C2H2指的DNA结合特异性和机制已经得到了深入的研究。另外，一个典型的C2H2指只识别三个或四个碱基对位点。这就提出了端粒六聚体TTAGGG DNA如何被TZAP特异性识别的问题。值得注意的是，在哺乳动物中，TZAP可以被归类为直接结合端粒重复DNA的一小部分蛋白质，包括庇护蛋白复合体的三个成员（TRF1、TRF2和单链DNA结合蛋白POT1）和HOT1[10,11]。TRF1、TRF2和HOT1使用同源结构域识别双链端粒DNA，不识别亚端粒DNA，而TZAP偏爱不同类型的亚端粒DNA[7,10,11]。与TRF1、TRF2和HOT1明确的DNA结合机制相反，TZAP与（亚）端粒DNA特异性相互作用的机制需要进一步研究。我们首先生成了一个包含Znf9-11（残基516-605）的TZAP结构。通过FP（荧光偏振）测定，我们发现Znf9-11构建体对含有TTAGGG序列的双链寡核苷酸（称为TTAGGG探针）具有相对较低的结合亲和力（bbb30µM）（图1B和补充信息，表S1）。然后我们注意到，在Znf11的c端有一个进化上保守且高度碱性的区域（残基606-620）（补充信息，图S1）。含有Znf9-11和保守的c -末端区域（516-620残基，简称Znf9-11- c）的构建体在150 mM NaCl溶液中以约0.18µM的KD与TTAGGG dsDNA探针结合，表明c -末端区域在端粒DNA结合中起关键作用（图1B）。
Telomere length homeostasis, dictating cellular proliferative potential, is crucial for proper cellular function. In addition to the well-known telomere shortening during cell division and lengthening by activation of telomerase or an alternative lengthening of telomeres (ALT) mechanism, telomere length is also subject to regulated rapid deletion events referred to as telomere trimming [1]. This trimming process involves excision of telomeric structures called T-loops, which requires homologous recombination proteins XRCC3 and NBS1 [2, 3]. Recent studies showed that the balance between telomere trimming and lengthening determines telomere length in germline and stem cells, suggesting that telomere trimming should be under stringent control [4, 5]. However, the regulation of telomere trimming remains largely unknown. Recently, two groups established human TZAP (ZBTB48, renamed telomeric zinc finger-associated protein) as a factor that directly binds double-stranded telomeric TTAGGG sequence and stimulates telomere trimming [6-9]. TZAP is composed of an N-terminal BTB/POZ domain and eleven adjacent C2H2-type zinc fingers (Znf1-11) at its C-terminus (Figure 1A). Li et al.[6] mapped TZAP’s TTAGGG binding region to Znf9-11, whereas further study refined it specifically to Znf11 [7]. This is the first time, to our knowledge, that a C2H2 finger was reported to bind telomeric DNA directly, although the DNA binding specificity and mechanisms of C2H2 finger have been intensely studied. In addition, one canonical C2H2 finger only recognizes three or four base pair sites. This raises the question of how the telomeric hexamer TTAGGG DNA is specifically recognized by TZAP. Notably, TZAP can be classified into a small subset of proteins that directly binds telomeric repeat DNA in mammals, including three members of the shelterin complex (TRF1, TRF2, and single-stranded DNA binding protein POT1) and HOT1 [10, 11]. TRF1, TRF2 and HOT1 employ homeodomains for double-stranded telomeric DNA recognition and do not recognize subtelomeric DNA, whereas TZAP has preference for distinct types of subtelomeric DNA [7, 10, 11]. In contrast to the well-defined DNA binding mechanisms of TRF1, TRF2 and HOT1, the mechanisms by which TZAP specifically interacts with (sub) telomeric DNA requires further investigation.We first generated a construct of TZAP that included Znf9-11 (residues 516-605). Using FP (fluorescence polarization) assay, we showed that the Znf9-11 construct displayed a relatively low binding affinity (> 30 µM) to a double-stranded oligonucleotide containing TTAGGG sequence (referred to as TTAGGG probe)(Figure 1B and Supplementary information, Table S1). We then noticed that there is an evolutionarily conserved and highly basic region (residues 606-620) located immediately C-terminal to Znf11 (Supplementary information, Figure S1). The construct containing Znf9-11 and the conserved C-terminal region (residues 516-620, referred to as Znf9-11-C) bound the TTAGGG dsDNA probe with a KD of about 0.18 µM in 150 mM NaCl solution, indicating a critical role of the C-terminal region in telomeric DNA binding (Figure 1B).