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从莱茵衣藻细胞中制备高效的具有切除修复能力的无细胞提取物。

Preparation of efficient excision repair competent cell-free extracts from C. reinhardtii cells.

作者信息

Chaudhari Vishalsingh, Raghavan Vandana, Rao Basuthkar J

机构信息

Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, India.

出版信息

PLoS One. 2014 Oct 9;9(10):e109160. doi: 10.1371/journal.pone.0109160. eCollection 2014.

DOI:10.1371/journal.pone.0109160
PMID:25299516
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4192114/
Abstract

Chlamydomonas reinhardtii is a prospective model system for understanding molecular mechanisms associated with DNA repair in plants and algae. To explore this possibility, we have developed an in vitro repair system from C. reinhardtii cell-free extracts that can efficiently repair UVC damage (Thymine-dimers) in the DNA. We observed that excision repair (ER) synthesis based nucleotide incorporation, specifically in UVC damaged supercoiled (SC) DNA, was followed by ligation of nicks. Photoreactivation efficiently competed out the ER in the presence of light. In addition, repair efficiency in cell-free extracts from ER deficient strains was several fold lower than that of wild-type cell extract. Interestingly, the inhibitor profile of repair DNA polymerase involved in C. reinhardtii in vitro ER system was akin to animal rather than plant DNA polymerase. The methodology to prepare repair competent cell-free extracts described in the current study can aid further molecular characterization of ER pathway in C. reinhardtii.

摘要

莱茵衣藻是用于理解植物和藻类中与DNA修复相关分子机制的一个有前景的模式系统。为了探索这种可能性，我们从莱茵衣藻无细胞提取物中开发了一种体外修复系统，该系统能够有效修复DNA中的紫外线C损伤（胸腺嘧啶二聚体）。我们观察到，基于切除修复（ER）合成的核苷酸掺入，特别是在紫外线C损伤的超螺旋（SC）DNA中，随后是切口的连接。在有光的情况下，光复活能有效地竞争掉ER。此外，ER缺陷菌株的无细胞提取物中的修复效率比野生型细胞提取物低几倍。有趣的是，莱茵衣藻体外ER系统中参与修复的DNA聚合酶的抑制剂谱类似于动物而非植物的DNA聚合酶。本研究中描述的制备具有修复能力的无细胞提取物的方法有助于进一步对莱茵衣藻中ER途径进行分子表征。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22d6/4192114/702c5cfed65e/pone.0109160.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22d6/4192114/fbbaa386eaf3/pone.0109160.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22d6/4192114/9f9f5667282c/pone.0109160.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22d6/4192114/308696444fff/pone.0109160.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22d6/4192114/f28293ca5329/pone.0109160.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22d6/4192114/702c5cfed65e/pone.0109160.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22d6/4192114/fbbaa386eaf3/pone.0109160.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22d6/4192114/9f9f5667282c/pone.0109160.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22d6/4192114/308696444fff/pone.0109160.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22d6/4192114/f28293ca5329/pone.0109160.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22d6/4192114/702c5cfed65e/pone.0109160.g005.jpg

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DNA 修复蛋白 XRCC1 通过刺激胞嘧啶甲基化（5-meC）切除、缺口修复和 DNA 连接，在植物 DNA 去甲基化途径中发挥作用。
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