Journal
DNA REPAIR
Volume 15, Issue -, Pages 21-28Publisher
ELSEVIER
DOI: 10.1016/j.dnarep.2013.12.008
Keywords
Translesion DNA synthesis; DNA polymerase kappa; Benzo[a]pyrene diolepoxide-N-2-guanine
Categories
Funding
- Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) [18201010, 22241016]
- Ministry of Health, Labour and Welfare, Japan (MHLW) [H21-Food-General-009]
- Japan Health Science Foundation [KHB1007]
- MHLW [20 designated-8]
- Food Safety Commission
- Grants-in-Aid for Scientific Research [24659035, 22241016] Funding Source: KAKEN
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Humans possess multiple specialized DNA polymerases that continue DNA replication beyond a variety of DNA lesions. DNA polymerase kappa (Pol kappa) bypasses benzo[a]pyrene diolepoxide-N-2-deoxyguanine (BPDE-N-2-dG) DNA adducts in an almost error-free manner. In the previous work, we changed the amino acids close to the adducts in the active site and examined the bypass efficiency. The substitution of alanine for phenylalanine 171 (F171A) enhanced by 18-fold in vitro, the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N-2-dG. In the present study, we established human cell lines that express wild-type Pol kappa (POLK+/-), F171A (POLK F171A/-) or lack expression of Pol kappa (POLK-/-) to examine the in vivo significance. These cell lines were generated with Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, which has high efficiency for gene targeting. Mutations were analyzed with shuttle vectors having (-)- or (+)-trans-anti-BPDE-N-2-dG in the supF gene. The frequencies of mutations were in the order of POLK-/- > POLK+/- F171A/- both in (-)- and (+)-trans-anti-BPDE-N-2-dG. These results suggest that F171 may function as a molecular brake for bypass across BPDE-N-2-dG by Pol K and raise the possibility that the cognate substrates for Pol K are not BP adducts in DNA but may be lesions in DNA induced by endogenous mutagens. (C) 2014 Elsevier B.V. All rights reserved.
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