Tandem lesions, comprised of two adjacent damaged nucleotides, make up a family of DNA damages formed by nucleobase radicals frequently generated by ionizing radiation. One such tandem lesion contains a thymine glycol (Tg) flanked on its 5’-side by a 2-deoxyribonolactone (L*). To study replication properties of this tandem lesion, we prepared three modified plasmid constructs containing L*Tg and single lesions Tg and L*, which were replicated in HEK 293T cells. After incubation for 2 days, progeny DNA was collected, purified, and used to transform E. coli DH10B cells, and the transformants were analyzed by oligonucleotide hybridization followed by DNA sequencing. While viability of Tg containing constructs was ~95% in this 5’-CTTgG-3’ sequence, suggesting that in this sequence context Tg is not a strong block of DNA replication in human cells, the L* lesion reduced the viability to &lt;20%. Viability was further decreased in the tandem lesion 5’-CL*TgG-3’ to ~6%. Analyses of the progeny from Tg showed that nearly 97% replication involved dA incorporation opposite Tg with 1.6% other nucleotide incorporations and 1.6% targeted single-base deletions. In contrast, the tandem L*Tgconstruct showed 19% one-base deletions. In the full-length products from L*Tg, ~80% and 88% dA incorporations occurred opposite L* and Tg, respectively. Therefore, Tg is nearly 4-times more mutagenic when it is 3’ to L*. To determine the roles of translesion synthesis (TLS) DNA polymerases,we examined the replication of these constructs in HEK 293T cells either with knockout in human DNA polymerase η (hPol η) expression, or with knockdown of hPol ζ or hPol ι. For the tandem lesion, the rate of one base deletions increased from 19% in the HEK 293T cells to 26% and 34%, respectively, in the hPol ζ and hPol ι knockdown cell lines; additionally, ~3% two-base deletions occurred in these pol knockdown cells. In contrast, neither the viability nor the deletion frequency was altered in hPol η knockout cells. This suggests that the TLS polymerases ζ and ι but not hpol η play a critical role in generating full-length products from replication of this tandem lesion. In the full-length products,dA incorporation also was lowered from 81% to 71% and 63%, respectively, in the hPol ζ and hPol ι deficient cells, suggesting that these polymerases are involved in dA incorporations opposite both L* and Tg. The biological implication of these results will be discussed.

Translesion synthesis of tandem DNA lesions containing 2-deoxyribonolactone 5’ to thymine glycol in human cells

2019 Fall National Meeting

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