Abstract

Purpose

DNA, the hereditary material of a human cell generally exists as Watson-Crick base paired double-stranded B-DNA. Studies suggest that DNA can also exist in non-B forms, such as four stranded G-quadruplexes (G4 DNA). Recently, our studies revealed that the regions of DNA that can fold into G-quadruplex structures are less sensitive to ionizing radiation (IR) compared to B-DNA. Importantly, we reported that the planar G-quartet of a G4 structure is shielded from radiation induced DNA breaks, while the single- and double-stranded DNA regions remained susceptible. Thus, in the present study, we investigate whether telomeric repeat DNA present at the end of telomere, known to fold into G4 DNA can protect from radiation induced damages including strand breaks, oxidation of purines and bulky adduct formation on DNA.

Materials and methods

For plasmid irradiation assay, plasmids containing human telomeric repeat DNA sequence TTAGGG (0.8 kb or 1.8 kb) were irradiated with increasing doses of IR along with appropriate control plasmids and products were resolved on 1% agarose gel. Radioprotection was evaluated based on extent of conversion of supercoiled to nicked or linear forms of the DNA following irradiation. Formation of G-quadruplex structure on supercoiled DNA was evaluated based on circular dichroism (CD) spectroscopy studies. Cleavage of radiation induced oxidative damage and extent of formation of nicks was further evaluated using base and nucleotide excision repair proteins.

Results

Results from CD studies showed that the plasmid DNA harboring human telomeric repeats (TTAGGG) can fold into G-quadruplex DNA structures. Further, results showed that human telomeric repeat sequence when present on a plasmid can protect the plasmid DNA against IR induced DNA strand breaks, unlike control plasmids bearing random DNA sequence.

Conclusions

Human telomeric repeat sequence when present on plasmids can fold into G-quadruplex DNA structures, and can protect the DNA against IR induced DNA strand breaks and oxidative damage. These results in conjunction with our previous studies suggest that telomeric repeat sequence imparts less sensitivity to IR and thus telomeres of chromosomes are protected from radiation.

中文翻译：

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人类端粒 DNA 中存在的 G4 DNA 有助于降低对 γ 辐射诱导的氧化损伤的敏感性，但不会形成大量加合物

 抽象的

 目的

DNA，人类细胞的遗传物质，通常以 Watson-Crick 碱基配对的双链 B-DNA 形式存在。研究表明，DNA 也可以以非 B 形式存在，例如四链 G-四链体 (G4 DNA)。最近，我们的研究表明，与 B-DNA 相比，可折叠成 G-四链体结构的 DNA 区域对电离辐射 (IR) 的敏感性较低。重要的是，我们报道了 G4 结构的平面 G 四联体免受辐射诱导的 DNA 断裂的影响，而单链和双链 DNA 区域仍然容易受到影响。因此，在本研究中，我们研究了存在于端粒末端的端粒重复DNA（已知折叠成G4 DNA）是否可以防止辐射引起的损伤，包括链断裂、嘌呤氧化和DNA上大体积加合物的形成。

 材料和方法

对于质粒辐照测定，用增加剂量的IR辐照含有人端粒重复DNA序列TTAGGG（0.8kb或1.8kb）的质粒以及适当的对照质粒，并在1%琼脂糖凝胶上解析产物。辐射防护根据辐射后DNA的超螺旋转化为切口或线性形式的程度进行评估。基于圆二色性 (CD) 光谱研究评估了超螺旋 DNA 上 G-四链体结构的形成。使用碱基和核苷酸切除修复蛋白进一步评估辐射诱导的氧化损伤的裂解和切口形成的程度。

 结果

CD 研究结果表明，含有人类端粒重复序列 (TTAGGG) 的质粒 DNA 可以折叠成 G-四链体 DNA 结构。此外，结果表明，与带有随机DNA序列的对照质粒不同，当存在于质粒上时，人端粒重复序列可以保护质粒DNA免受IR诱导的DNA链断裂。

 结论

人端粒重复序列存在于质粒上时可以折叠成 G-四链体 DNA 结构，并可以保护 DNA 免受 IR 诱导的 DNA 链断裂和氧化损伤。这些结果与我们之前的研究相结合表明，端粒重复序列对 IR 的敏感性较低，因此染色体端粒受到保护免受辐射。