DNA and Chromosomes
Human DNA polymerase η has reverse transcriptase activity in cellular environments
Classical DNA and RNA polymerase (pol) enzymes have defined roles with their respective substrates, but several pols have been found to have multiple functions. We reported previously that purified human DNA pol η (hpol η) can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) and can use both DNA and RNA as substrates. X-ray crystal structures revealed that two pol η residues, Phe-18 and Tyr-92, behave as steric gates to influence sugar selectivity.Human DNA polymerase η accommodates RNA for strand extension
Ribonucleotides are the natural analogs of deoxyribonucleotides, which can be misinserted by DNA polymerases, leading to the most abundant DNA lesions in genomes. During replication, DNA polymerases tolerate patches of ribonucleotides on the parental strands to different extents. The majority of human DNA polymerases have been reported to misinsert ribonucleotides into genomes. However, only PrimPol, DNA polymerase α, telomerase, and the mitochondrial human DNA polymerase (hpol) γ have been shown to tolerate an entire RNA strand.Structural and Kinetic Analysis of Miscoding Opposite the DNA Adduct 1,N6-Ethenodeoxyadenosine by Human Translesion DNA Polymerase η
1,N6-Ethenodeoxyadenosine (1,N6-ϵdA) is the major etheno lesion formed in the reaction of DNA with epoxides substituted with good leaving groups (e.g. vinyl chloride epoxide). This lesion is also formed endogenously in DNA from lipid oxidation. Recombinant human DNA polymerase η (hpol η) can replicate oligonucleotide templates containing 1,N6-ϵdA. In steady-state kinetic analysis, hpol η preferred to incorporate dATP and dGTP, compared with dTTP. Mass spectral analysis of incorporation products also showed preferred purine (A, G) incorporation and extensive −1 frameshifts, suggesting pairing of the inserted purine and slippage before further replication.Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η
Ribonucleotides and 2′-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2′-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2′-OH group.Roles of Residues Arg-61 and Gln-38 of Human DNA Polymerase η in Bypass of Deoxyguanosine and 7,8-Dihydro-8-oxo-2′-deoxyguanosine
Background: Arg-61 and Gln-38 of human DNA polymerase (hpol) η play important roles in the catalytic reaction.Results: Mutations R61M or Q38A/R61A dramatically disrupt the activity of hpol η.Conclusion: Polarized water molecules can mimic and partially compensate for the missing side chains of Arg-61 and Gln-38 in the Q38A/R61A mutant.Significance: The positioning and positive charge of Arg-61 synergistically contribute to the activity of hpol η, with additional effects of Gln-38.Structural and Kinetic Analysis of Nucleoside Triphosphate Incorporation Opposite an Abasic Site by Human Translesion DNA Polymerase η
Background: Abasic sites are the most common lesion in DNA.Results: Kinetic and mass spectrometric assays demonstrate that human polymerase (pol) η preferentially inserts A and G opposite an abasic site.Conclusion: Crystal structures reveal H-bonding between incoming ATP and GTP and the 5′-phosphate of the abasic moiety.Significance: Abasic site bypass by pol η follows a “purine rule” for insertion, with formation of frameshifts.
