DNA polymerase κ participates in early S-phase DNA replication in human cells

Abstract

Cycling cells replicate their DNA during the S phase through a defined temporal program known as replication timing. Mutation frequencies, epigenetic chromatin states, and transcriptional activities are different for genomic regions that are replicated early and late in the S phase. Here, we found from ChIP-Seq analysis that DNA polymerase (Pol) κ is enriched in early-replicating genomic regions in HEK293T cells. In addition, by feeding cells with N ²-heptynyl-2'-deoxyguanosine followed by click chemistry-based enrichment and high-throughput sequencing, we observed elevated Pol κ activities in genomic regions that are replicated early in the S phase. On the basis of the established functions of Pol κ in accurate and efficient nucleotide insertion opposite endogenously induced N ²-modified dG lesions, our work suggests that active engagement of Pol κ may contribute to diminished mutation rates observed in early-replicating regions of the human genome, including cancer genomes. Together, our work expands the functions of Pol κ and offered a plausible mechanism underlying replication timing-dependent mutation accrual in the human genome.

Keywords: DNA replication; mutagenesis; replication timing; translesion synthesis polymerase.

Fig. 1.

CRISPR knock-in of three tandem repeats of Flag tag to the C terminus of endogenous Pol κ in HEK293T cells and N²-hep-dG-Seq. (A) Agarose gel electrophoresis for detecting insertion of 3×Flag to the C terminus of endogenous Pol κ in HEK293T cells. PCR primers were designed to target outside the homology arms and yield a longer PCR product if the tag is inserted, where the expected amplicon lengths of parental HEK293T cells and the knock-in clone are 424 and 601 bp, respectively. (B) Western blot with anti-Flag and Pol κ antibodies for confirming the successful knock-in (KI) of Flag tag to endogenous Pol κ protein, where Pol κ-Flag is expressed at a lower level than the untagged counterpart. (C) The chemical structure of N²-hep-dG. (D and E) Frequencies of N²-hep-dG in cellular DNA isolated from asynchronized HEK293T cells, the isogenic POLK^-−/− cells, and the POLK^−/− cells transfected with an empty vector or a vector for expressing C-terminally Flag-tagged human Pol κ (D), as well as from synchronized S-phase and non-S-phase HEK293T cells (E). (F) Denaturing gel electrophoresis showing the reaction of a 12-mer oligodeoxynucleotide, d(ATGGCGXGCTAT) (X = N²-hep-dG), with biotin-azide. (G) Dot blot monitoring the conjugation of biotin-azide with genomic DNA isolated from HEK293T cells fed with N²-hep-dG. (H) N²-hep-dG-Seq enables efficient enrichment of a 90-mer N²-hep-dG-containing duplex DNA over the corresponding unmodified DNA. The enrichment fold was calculated by normalizing the qPCR-determined recovery rates of N²-hep-dG-containing DNA over the unmodified counterpart. The data in (D), (E), and (H) represent the mean ± SD of results obtained from three independent experiments. ***P < 0.001; ****P < 0.0001 (unpaired two-tailed Student’s t test).

Fig. 2.

N²-hep-dG and polymerase κ are enriched at genomic regions replicated in the early S phase of the cell cycle. (A) Spearman’s correlation coefficients of replication timing (PRJNA419407), Pol κ ChIP-seq, and N²-hep-dG-Seq. (B) Representative IGV plots illustrating the comparisons of replication timing, Pol κ ChIP-seq, and N²-hep-dG-Seq results for chromosomes 7, 11, and 12. The bin size for generating the IGV plots in (B–D) was 50 kb.