Akua Tzofit, Rahav Galia, Saragani Yossi, Hizi Amnon, Bakhanashvili Mary
aInfectious Diseases Unit, Sheba Medical Center, Tel HashomerbDepartment of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel AvivcMina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.
AIDS. 2017 Jan 28;31(3):343-353. doi: 10.1097/QAD.0000000000001339.
OBJECTIVE(S): HIV-1 reverse transcriptase frequently incorporates ribonucleotides into the proviral DNA in macrophages, but not in lymphocytes. The enzyme exerts an efficient ribonucleotide-terminated primer extension capacity. Furthermore, ribonucleotide-editing repair is attenuated in macrophages. Tumor suppressor p53 protein, displaying an intrinsic 3'→5' exonuclease activity, was found to be involved in efficient proofreading of base-base mismatches produced during DNA synthesis. As the presence of proofreading activity is cardinal for the DNA synthesis accuracy, it was of interest to assess whether p53 can serve as a trans-acting proofreader for HIV-1 reverse transcriptase during ribonucleotide incorporation.
We investigated the potential involvement of cytoplasmic p53 in error correction during insertion of ribonucleotides into DNA by recombinant HIV-1 reverse transcriptase in a p53-proficient and deficient background.
Primer extension reactions were carried out to elucidate the incorporation and removal of ribonucleotides.
The biochemical studies suggest that p53 is involved in a ribonucleotide damage-associated repair mechanism through its capacity to remove preformed 3'-terminal ribonucleotides, to decrease ribonucleotide incorporation and to prevent the 3'-ribo-terminated primer extension during ongoing DNA synthesis by HIV-1 reverse transcriptase. A positive correlation exists between the presence of endogenous p53 and decrease in stable incorporation of ribonucleotides into DNA with p53-harboring lysates of HCT116 cells. p53, by preferential removal of purine over pyrimidine ribonucleotides, may affect the ribonucleotide mutation spectra produced by HIV-1 reverse transcriptase.
The data implies that p53 can excise incorrect sugar in addition to base mispairs, thereby expanding the role of p53 in the repair of nucleic acids replication errors.
HIV-1逆转录酶经常在巨噬细胞而非淋巴细胞的前病毒DNA中掺入核糖核苷酸。该酶具有高效的核糖核苷酸终止引物延伸能力。此外，巨噬细胞中的核糖核苷酸编辑修复功能减弱。肿瘤抑制蛋白p53具有内在的3'→5'核酸外切酶活性，被发现参与DNA合成过程中产生的碱基错配的有效校对。由于校对活性的存在对DNA合成准确性至关重要，因此有必要评估p53在核糖核苷酸掺入过程中是否可作为HIV-1逆转录酶的反式作用校对酶。
我们在p53功能正常和缺陷的背景下，研究了细胞质p53在重组HIV-1逆转录酶将核糖核苷酸插入DNA过程中的纠错潜在作用。
进行引物延伸反应以阐明核糖核苷酸的掺入和去除情况。
生化研究表明，p53通过其去除预先形成的3'-末端核糖核苷酸、减少核糖核苷酸掺入以及在HIV-1逆转录酶进行DNA合成过程中防止3'-核糖末端引物延伸的能力，参与了与核糖核苷酸损伤相关的修复机制。HCT116细胞含有p53的裂解物中，内源性p53的存在与核糖核苷酸稳定掺入DNA的减少之间存在正相关。p53通过优先去除嘌呤核糖核苷酸而非嘧啶核糖核苷酸，可能会影响HIV-1逆转录酶产生的核糖核苷酸突变谱。
数据表明，p53除了能切除碱基错配外，还能切除错误的糖，从而扩展了p53在核酸复制错误修复中的作用。
