Repetitive DNA sequences support the formation of structures that can interrupt replication and repair, leading to breaks and mutagenesis. One particularly stable structure is G-quadruplex (G4) DNA, which is four-stranded and formed from tandemly repetitive guanine bases. When folded within a template, G4 interferes with DNA synthesis. Similar to non-duplex structures, DNA base lesions can also halt an advancing replication fork, but the Y-family polymerases solve this problem by bypassing the damage. In order to better understand how guanine-rich DNA is replicated, we have investigated the activity of the model Y-family polymerase, Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), on guanine-rich templates in vitro. We find that Dpo4 progression on templates containing either a single GC-rich hairpin or a G4 DNA structure is greatly reduced and synthesis stalls at the structure. Human polymerase eta (hPol eta) showed the same pattern of stalling at G4; however, and in contrast to Klenow, hPol eta and Dpo4 partially synthesise into the guanine repeat. Substitution of the nucleotide selectivity residue in Dpo4 with alanine permitted ribonucleotide incorporation on unstructured templates, but this further reduced the ability of Dpo4 to synthesise across from the guanine repeats. The advancement of Dpo4 on G4 templates was highest when the reaction was supplied with only deoxycytidine triphosphate, suggesting that high-fidelity synthesis is favoured over misincorporation. Our results are consistent with a model where the Y-family polymerases pause upon encountering G4 structures but have an ability to negotiate some synthesis through tetrad-associated guanines. This suggests that the Y-family polymerases reduce mutagenesis by catalysing the accurate replication of repetitive DNA sequences, but most likely in concert with additional replication and structure resolution activities.

中文翻译：

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硫枯草菌Dpo4聚合酶在体外对DNA G-四链体的反应。

重复的DNA序列支持可中断复制和修复，导致断裂和诱变的结构的形成。一种特别稳定的结构是G-四链体（G4）DNA，它是四链的，由串联重复的鸟嘌呤碱基形成。当折叠在模板中时，G4会干扰DNA合成。与非双链体结构相似，DNA碱基损伤也可以阻止前进的复制叉，但是Y家族聚合酶通过绕过损伤来解决此问题。为了更好地了解富鸟嘌呤的DNA是如何复制的，我们在富鸟嘌呤的模板上研究了Y型家族聚合酶Sulfolobus solfataricus P2 DNA聚合酶IV（Dpo4）的活性。我们发现，包含单个富含GC的发夹或G4 DNA结构的模板上的Dpo4进程大大减少，合成停滞在该结构上。人聚合酶eta（hPol eta）在G4处显示出相同的停顿模式；但是，与Klenow相反，hPol eta和Dpo4部分合成鸟嘌呤重复序列。用丙氨酸替代Dpo4中的核苷酸选择性残基，可将核糖核苷酸掺入非结构化模板中，但这进一步降低了Dpo4从鸟嘌呤重复序列合成的能力。当仅用脱氧胞苷三磷酸提供反应时，Dpo4在G4模板上的进展最高，这表明高保真合成优于错误掺入。我们的结果与一个模型一致，在该模型中，Y族聚合酶在遇到G4结构时会暂停，但具有通过四联体鸟嘌呤来协商某些合成的能力。这表明Y族聚合酶通过催化重复DNA序列的精确复制来减少诱变，但最有可能与其他复制和结构解析活性相一致。