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

Vishalsingh Chaudhari,Basuthkar J Rao,Vandana Raghavan,Yanbin Zhang

doi:10.1371/journal.pone.0109160

Vishalsingh Chaudhari, Basuthkar J Rao + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0109160

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Journal: PloS one	Publication Date: Oct 9, 2014
Citations: 3	License type: CC BY 4.0

Affiliation: Tata Institute of Fundamental Research

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.

Highlights

Genomic DNA of all organisms is constantly exposed to various endogenous and exogenous damaging agents
Bulky lesions are the major kind of UVC induced DNA damage typified by thymine dimers (TDs), which include both the cyclobutane pyrimidine dimers (CPDs) and less frequent pyrimidine (6–4) pyrimidone dimers (6–4 PPs) [1,2,3]
To assess whether the cellular system in the experimental conditions employed here exhibits robust enough excision of TDs, in vivo, we carried out the following experiment

Summary

Introduction

Genomic DNA of all organisms is constantly exposed to various endogenous and exogenous damaging agents. The current study describes the first attempt of demonstrating cell-free extract preparation from C. reinhardtii that is proficient in repairing UV induced TDs in plasmid DNA substrates.

Results

Conclusion