In vivo evidence that phenylalanine 171 acts as a molecular brake for translesion DNA synthesis across benzo[a]pyrene DNA adducts by human DNA polymerase κ

DNA Repair (Amst). 2014 Mar;15:21-8. doi: 10.1016/j.dnarep.2013.12.008. Epub 2014 Jan 21.

Abstract

Humans possess multiple specialized DNA polymerases that continue DNA replication beyond a variety of DNA lesions. DNA polymerase kappa (Pol κ) 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 κ (POLK+/-), F171A (POLK F171A/-) or lack expression of Pol κ (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+/->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 κ and raise the possibility that the cognate substrates for Pol κ are not BP adducts in DNA but may be lesions in DNA induced by endogenous mutagens.

Keywords: Benzo[a]pyrene diolepoxide-N(2)-guanine; DNA polymerase κ; Translesion DNA synthesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / analogs & derivatives*
  • Amino Acid Substitution
  • Base Sequence
  • Catalytic Domain
  • Cell Line
  • DNA Damage
  • DNA Repair*
  • DNA Replication*
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics*
  • DNA-Directed DNA Polymerase / metabolism
  • Deoxyguanosine / analogs & derivatives*
  • Deoxyguanosine / genetics
  • Humans
  • Mutagenesis, Site-Directed
  • Mutation Rate
  • Phenylalanine / genetics

Substances

  • Phenylalanine
  • 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide
  • 7,8-dihydroxy-9,10-epoxide-7,8,9,10-tetrahydrobenzo(a)pyrene-10-deoxyguanosine
  • DNA-Directed DNA Polymerase
  • POLK protein, human
  • Deoxyguanosine