title: Pol kappa partially rescues MMR-dependent cytotoxicity of O6-methylguanine.
abstract: To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous sources of DNA damage. DNA integrity is maintained by the coordinated action of DNA damage response mechanisms and DNA repair. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage but are potentially error-prone. Here, we investigate the role of DNA polymerase κ (pol κ) in TLS across alkylated lesions by silencing this polymerase (pol) in human cells using transient small RNA interference. We show that human pol κ has a significant protective role against methyl nitrosourea (MNU)-associated cytotoxicity without affecting significantly mutagenicity. The increase in MNU-induced cytotoxicity when pol κ is down-regulated was affected by the levels of O6-methylguanine DNA methyltransferase and fully abolished when mismatch repair (MMR) was defective. Following MNU treatment, the cell cycle profile was unaffected by the pol κ status. The downregulation of pol κ caused a severe delay in the onset of the second mitosis that was fully dependent on the presence of O6-methylguanine ( O6-meGua) lesions. After MNU exposure, in the absence of pol κ, the frequency of sister chromatid exchanges was unaffected whereas the induction of RAD 51 foci increased. We propose that pol κ partially protects human cells from the MMR-dependent cytotoxicity of O6-meGua lesions by restoring the integrity of replicated duplexes containing single-stranded gaps generated opposite O6-meGua facilitated by RAD 51 binding.
authors.affiliation: Department of Environment and Primary Prevention, Section of Molecular Epidemiology, Rome, Italy.
authors.forename: Eliana
authors.initials: E
authors.lastname: Lupari
authors.forename: Ilenia
authors.initials: I
authors.lastname: Ventura
authors.forename: Francesca
authors.initials: F
authors.lastname: Marcon
authors.forename: Gabriele
authors.initials: G
authors.lastname: Aquilina
authors.forename: Eugenia
authors.initials: E
authors.lastname: Dogliotti
authors.forename: Paola
authors.initials: P
authors.lastname: Fortini
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D053843:DNA Mismatch Repair; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008770:Methylnitrosourea / Q000494:pharmacology; D008938:Mitosis / Q000187:drug effects; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D034622:RNA Interference; D051135:Rad51 Recombinase / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012854:Sister Chromatid Exchange / Q000187:drug effects; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
chemical_list: D000477:Alkylating Agents; D004259:DNA-Directed DNA Polymerase; D006147:Guanine; D008770:Methylnitrosourea; D019853:O(6)-Methylguanine-DNA Methyltransferase; D018809:Proliferating Cell Nuclear Antigen; D051135:Rad51 Recombinase; C008449:O-(6)-methylguanine; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases