浙江大学博士生连发Nature，PNAS文章

　　

   浙江大学与美国国立卫生研究院（NIH）的研究人员继2010年首次解析人类DNA聚合酶η 的分子结构之后，于近期分别在DNA聚合酶的催化反应机制以及对顺铂药物的拮抗作用研究方面获得了突破性的进展——揭示了新的催化反应特点，以及独特的抑制剂结合位点。相关成果分别公布在国际期刊Nature与PNAS杂志上。

   浙江大学的生物物理学博士研究生赵烨为主要完成人之一，指导老师为浙江大学华跃进教授和美国NIH的杨薇研究员。至此，赵烨在其攻读博士论文期间以第一或共同第一作者在Nature发表论文二篇、PNAS一篇。

   DNA经常受到内源和外界环境因子的攻击，从而造成DNA的损伤。其中大部分可以被无误的DNA修复机制所识并修复，但也有一些会因为在复制过程中没有被修复而在染色体上继续存在，造成复制叉的停顿，从而影响细胞的分裂，更加严重的会导致细胞的死亡。

   为了应对这些损伤，细胞进化产生了一种损伤耐受机制，DNA聚合酶η 就是其中一种重要的损伤合成酶。分子结构显示人类DNA聚合酶η具有一个能够完美容纳损伤嘧啶二聚体的活性中心以及分子夹板的功能。而另外一方面，强大的跨损伤合成活性也使其对于癌症治疗药物顺铂具有一定的拮抗作用。

   在第一篇题为“Structural basis of human DNA polymerase η-mediated chemoresistance to cisplatin”的文章中，研究人员首先通过体外延伸及稳态动力学研究，发现人类DNA聚合酶η只能够在顺铂交联的损伤DNA处进行正确的插入而无法有效的进行延伸，这意味着很有可能需要其他的DNA聚合酶接手去完成整个跨损伤合成过程。同时通过结构生物学的进一步分析也对各个步骤的合成效率进行了系统的解释。

   有趣的是在结构解析的过程中，研究者发现了一个对于人类DNA聚合酶η来说可能的特异性抑制剂结合位点。该位点通过与W297残基以类似于碱基堆积的方式相互作用，并且得到了体外实验的验证--高浓度的dATP对于 聚合酶的合成效率有很强的抑制效果。这一发现也为抗癌药物的研发提供了新的思路。

   尽管很久以前人们就对DNA聚合酶如何进行化学反应有所了解，但由于研究手段的局限性，具体的反应过程仍然是一个相对未知的领域。在第二篇题为“Watching DNA polymerase η make a phosphodiester bond”的文章中，研究人员巧妙的利用晶体内聚合酶反应速率减弱的特性，首次在晶体内观测到了完整的DNA聚合酶化学反应过程。

   研究清晰的描述了反应顺序首先为引物链的3’末端羟基去质子化，紧接着末端的脱氧核糖构象由C2’-endo变为C3’-endo并最终完成对于dATP的亲核攻击，形成新的磷酸二酯键。同时在反应过程中第一次观察到了第三个镁离子的出现，其作用很有可能是中和化学反应所产生的大量负电荷，稳定反应的中间体。这些发现大大提高了人们对于双向金属离子介导的核苷酸转移机制的认识。

（生物通）

原文摘要：

Structural basis of human DNA polymerase η-mediated chemoresistance to cisplatin

Cisplatin (cis-diamminedichloroplatinum) and related compounds cause DNA damage and are widely used as anticancer agents. Chemoresistance to cisplatin treatment is due in part to translesion synthesis by human DNA polymerase η (hPol η). Here, we report crystal structures of hPol η complexed with intrastrand cisplatin- 1,2–cross-linked DNA, representing four consecutive steps in translesion synthesis. In contrast to the generally enlarged and nondiscriminating active site of Y-family polymerases like Dpo4, Pol η is specialized for efficient bypass of UV–cross-linked pyrimidine dimers. Human Pol η differs from the yeast homolog in its binding of DNA template. To incorporate deoxycytidine opposite cisplatin crosslinked guanines, hPol η undergoes a specific backbone rearrangement to accommodate the larger base dimer and minimizes the DNA distortion around the lesion. Our structural analyses show why Pol η is inefficient at extending primers after cisplatin lesions, which necessitates a second translesion DNA polymerase to complete bypass in vivo. A hydrophobic pocket near the primer binding site in human Pol η is identified as a potential drug target for inhibiting translesion synthesis and, thereby, reducing chemoresistance.

Watching DNA polymerase η make a phosphodiester bond
DNA synthesis has been extensively studied, but the chemical reaction itself has not been visualized. Here we follow the course of phosphodiester bond formation using time-resolved X-ray crystallography. Native human DNA polymerase η, DNA and dATP were co-crystallized at pH 6.0 without Mg2+. The polymerization reaction was initiated by exposing crystals to 1 mM Mg2+ at pH 7.0, and stopped by freezing at desired time points for structural analysis. The substrates and two Mg2+ ions are aligned within 40 s, but the bond formation is not evident until 80 s. From 80 to 300 s structures show a mixture of decreasing substrate and increasing product of the nucleotidyl-transfer reaction. Transient electron densities indicate that deprotonation and an accompanying C2’-endo to C3’-endo conversion of the nucleophile 3’-OH are rate limiting. A third Mg21 ion, which arrives with the new bond and stabilizes the intermediate state, may be an unappreciated feature of the two-metal-ion mechanism.

Structure and mechanism of human DNA polymerase η
The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase η (Polη), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Polη at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Polη acts like a ‘molecular splint’ to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Polη orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Polη missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Polη in replicating through D loop and DNA fragile sites.