New Alternative Repair Pathways for Complex DNA Damage. Applications to the Mechanisms of Resistance to Anticancer Therapies.

Project: Research project

Grant Program

ORAU Grant

Project Description

We studied the enzymatic activities of human and bacterial repair enzymes including human APE1, NEIL1, NEIL3 were tested on P32 radioactively labeled synthetic oligonucleotides containing various chemical modifications of DNA bases. Our research produced new results, published in the professional journal Scientific Reports. Two young researchers from our team trained at DNA Repair lab at Gustave Rossi Cancer Campus, Villejuif, France. In 2017 we perform:
Calendar plan item 1: Construction of the plasmid vectors, expression and purification of the various DNA repair proteins APE1, NEIL1, NEIL2, NEIL3. Expression and purification of human Ape1 with E.coli strains Rozetta and BH110 DE3. Construction of expression vector POZ-C with genes NEIL1 and NEIL3 with flag and HA tags for expression in mammalian cells. Vector POZ-C, having NEIL1 and NEIL3 genes with flag and HA tags, was cloned in cell lines Phoenix to build retrovirus for following transfection into cancer cells. Creation of stable cancer cell lines Hela S3 with genes NEIL1 and NEIL3 with flag and HA tags. Selection of NEIL1 and NEIL3 from Hela S3 and Phoenix cancer cell lines.
Calendar plan item 2. Preparation of oligonucleotides with chemical and/or conformational modifications, construction and characterization of three- and four-stranded DNA structures containing a single psoralen-derived interstrand DNA cross-link.
Calendar plan item 3. Biochemical analysis (Redox, BER, NIR) of APE1 activity with radioactively labelled substrates with various damages was performed. The enzymatic activities of human and bacterial repair enzymes including human APE1, NEIL1, NEIL3 were qualitative and quantitatively characterized:
Calendar plan item 4. Analysis of nucleotide and amino acid sequences and protein structures, including sequence alignment of PARP proteins and short time molecular dynamics on DNA repair proteins.
Short titleNew Alternative Repair Pathways for Complex DNA Damage
AcronymCROSSLINK
StatusActive
Effective start/end date9/11/1712/31/20

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DNA Damage
DNA Repair
Chromatin
Radio
DNA
Neoplasms
DNA Breaks
Radiotherapy
Substrate Specificity
Epigenomics
Drug Therapy
Nucleic Acids
DNA Adducts
Therapeutics
Histone Code
Combination Drug Therapy
Research
Antineoplastic Agents
Adenosine Diphosphate Ribose
Lung Neoplasms