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The Role of DNA Polymerase Nu and Zeta in the Repair of DNA Interstrand Crosslinks
Author(s) -
Bernard Faviola,
Althaus Irene,
Fisher Adam,
Jasty Vijay,
Canman Christine
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.996.4
Subject(s) - dna polymerase , polymerase , dna repair , dna , microbiology and biotechnology , dna replication , biology , genome instability , dna polymerase ii , homologous recombination , nucleotide excision repair , dna damage , chemistry , rna , reverse transcriptase , biochemistry , gene
Interstrand DNA Cross‐links (ICLs) are covalent bonds between the two DNA strands. They can be induced by endogenous aldehydes produced by metabolic pathways or exogenous agents such as chemotherapeutic drugs. ICLs inhibit DNA replication and RNA transcription and are lethal if not repaired. Lesions that are not repaired may cause chromosome breakage and genome instability that could ultimately promote cancer. The Fanconi anaemia (FA) pathway is in charge of removing the ICLs by recruiting specific endonucleases to ‘unhook’ the ICL and DNA translesion synthesis polymerases (TLS) that insert and extend nucleotides opposite the unhooked ICL. Ultimately, the FA pathway directs homologous recombination repair enzymes to complete the repair process and restore the DNA to its original double stranded form. Our major goal is to identify which proteins of the FA pathway regulate TLS polymerase activity. We are currently investigating the roles DNA polymerase Nu (POLN) and DNA polymerase Zeta (REV3) play in TLS during ICL repair. Current models suggest that REV3 is required for TLS but it still unknown if POLN is also involved in the same pathway. Using RNA‐Interference, we depleted HeLa cells of POLN, REV3, or both proteins to determine if they act in the same pathway. First, we determined whether HeLa cells depleted of one or both DNA polymerases induced the similar or enhanced cytotoxicity as measured by colony forming assays following exposure to Cisplatin (CDDP) or Mitomycin‐C (MMC). Second, we examined whether double knockdown cells showed similar or elevated chromosomal aberrations following drug exposure. Third, we performed an immunofluorescence in which we stain for 53BP1 and p‐ATM, markers for DNA double strands breaks. Our preliminary data shows evidence of failure to complete ICL repair when REV3 or POLN are depleted from the cells. Together, the data suggests POLN and REV3 are involved in ICL repair.