TY - JOUR
T1 - Ribonucleotide incorporation by yeast DNA polymerase ζ
AU - Makarova, Alena V.
AU - McElhinny, Stephanie A.Nick
AU - Watts, Brian E.
AU - Kunkel, Thomas A.
AU - Burgers, Peter M.
N1 - Funding Information:
The authors thank Carrie Stith for help with protein purification. This work was supported in part by Grant GM032431 from the National Institutes of Health to P.M.B. and by Project Z01 ES065089 from the Division of Intramural Research of the National Institutes of Health, National Institute on Environmental Health Sciences, to T.A.K.
PY - 2014/6
Y1 - 2014/6
N2 - During replication in yeast, the three B family DNA replicases frequently incorporate ribonucleotides (rNMPs) into DNA, and their presence in the nuclear genome can affect genome stability. This prompted us to examine ribonucleotide incorporation by the fourth B family member, Pol ζ, the enzyme responsible for the majority of damage-induced mutagenesis in eukaryotes. We first show that Pol ζ inserts rNMPs into DNA and can extend primer termini containing 3'-ribonucleotides. We then measure rNMP incorporation by Pol ζ in the presence of its cofactors, RPA, RFC and PCNA and at normal cellular dNTP and rNTP concentrations that exist under unstressed conditions. Under these conditions, Pol ζ stably incorporates one rNMP for every 200-300 dNMPs incorporated, a frequency that is slightly higher than for the high fidelity replicative DNA polymerases. Under damage-induced conditions wherein cellular dNTP concentrations are elevated 5-fold, Pol ζ only incorporates one rNMP per 1300 dNMPs. Functional interaction of Pol ζ with the mutasome assembly factor Rev1 gives comparable rNMP incorporation frequencies. These results suggest that ribonucleotide incorporation into DNA during Pol ζ-mediated mutagenesis in vivo may be rare.
AB - During replication in yeast, the three B family DNA replicases frequently incorporate ribonucleotides (rNMPs) into DNA, and their presence in the nuclear genome can affect genome stability. This prompted us to examine ribonucleotide incorporation by the fourth B family member, Pol ζ, the enzyme responsible for the majority of damage-induced mutagenesis in eukaryotes. We first show that Pol ζ inserts rNMPs into DNA and can extend primer termini containing 3'-ribonucleotides. We then measure rNMP incorporation by Pol ζ in the presence of its cofactors, RPA, RFC and PCNA and at normal cellular dNTP and rNTP concentrations that exist under unstressed conditions. Under these conditions, Pol ζ stably incorporates one rNMP for every 200-300 dNMPs incorporated, a frequency that is slightly higher than for the high fidelity replicative DNA polymerases. Under damage-induced conditions wherein cellular dNTP concentrations are elevated 5-fold, Pol ζ only incorporates one rNMP per 1300 dNMPs. Functional interaction of Pol ζ with the mutasome assembly factor Rev1 gives comparable rNMP incorporation frequencies. These results suggest that ribonucleotide incorporation into DNA during Pol ζ-mediated mutagenesis in vivo may be rare.
KW - DNA polymerase
KW - Mutagenesis
KW - Ribonucleotides
KW - Translesion synthesis
UR - http://www.scopus.com/inward/record.url?scp=84900340576&partnerID=8YFLogxK
U2 - 10.1016/j.dnarep.2014.02.017
DO - 10.1016/j.dnarep.2014.02.017
M3 - Article
C2 - 24674899
AN - SCOPUS:84900340576
SN - 1568-7864
VL - 18
SP - 63
EP - 67
JO - DNA Repair
JF - DNA Repair
IS - 1
ER -