Abstract5-Formylcytosine (5fC) is an epigenetic DNA modification introduced via TET protein-mediated oxidation of 5-methyl-dC. We recently reported that 5fC form reversible DNA–protein conjugates (DPCs) with histone proteins in living cells (Ji et al. (2017) Angew. Chem. Int. Ed., 56:14130–14134). We now examined the effects of 5fC mediated DPCs on DNA replication. Synthetic DNA duplexes containing site-specific DPCs between 5fC and lysine-containing proteins and peptides were subjected to primer extension experiments in the presence of human translesion synthesis DNA polymerases η and κ. We found that DPCs containing histones H2A or H4 completely inhibited DNA replication, but the replication block was removed when the proteins were subjected to proteolytic digestion. Cross-links to 11-mer or 31-mer peptides were bypassed by both polymerases in an error-prone manner, inducing targeted C→T transitions and –1 deletions. Similar types of mutations were observed when plasmids containing 5fC-peptide cross-links were replicated in human embryonic kidney (HEK) 293T cells. Molecular simulations of the 11-mer peptide-dC cross-links bound to human polymerases η and κ revealed that the peptide fits well on the DNA major groove side, and the modified dC forms a stable mismatch with incoming dATP via wobble base pairing in the polymerase active site.
Edited by Patrick Sung DNA-protein cross-links can interfere with chromatin architecture, block DNA replication and transcription, and interfere with DNA repair. Here we synthesized a DNA 23-mer containing a sitespecific DNA-peptide cross-link (DpC) by cross-linking an 11-mer peptide to the DNA epigenetic mark 5-formylcytosine in synthetic DNA and used it to generate a DpC-containing plasmid construct. Upon replication of the DpC-containing plasmid in HEK 293T cells, approximately 9% of progeny plasmids contained targeted mutations and 5% semitargeted mutations. Targeted mutations included C3 T transitions and C deletions, whereas semitargeted mutations included several base substitutions and deletions near the DpC lesion. To identify DNA polymerases involved in DpC bypass, we comparatively studied translesion synthesis (TLS) efficiency and mutagenesis of the DpC in a series of cell lines with TLS polymerase knockouts or knockdowns. Knockdown of either hPolor hPol reduced the mutation frequency by nearly 50%. However, the most significant reduction in mutation frequency (50%-70%) was observed upon simultaneous knockout of hPol and hPol with knockdown of hPol , suggesting that these TLS polymerases play a critical role in error-prone DpC bypass. Because TLS efficiency of the DpC construct was not significantly affected in TLS polymerase-deficient cells, we examined a possible role of replicative DNA polymerases in their bypass and determined that hPol ␦ and hPol ⑀ can accurately bypass the DpC. We conclude that both replicative and TLS polymerases can bypass this DpC lesion in human cells but that mutations are induced mainly by TLS polymerases.
Breast cancers are known to be driven by the transcription factor estrogen receptor and its ligand estrogen. While the receptor's cis-binding elements are known to vary between tumors, heterogeneity of hormone signaling at a single-cell level is unknown. In this study, we systematically tracked estrogen response across time at a single-cell level in multiple cell line and organoid models. To accurately model these changes, we developed a computational tool (TITAN) that quantifies signaling gradients in single-cell datasets. Using this approach, we found that gene expression response to estrogen is non-uniform, with distinct cell groups expressing divergent transcriptional networks. Pathway analysis suggested the two most distinct signatures are driven separately by ER and FOXM1. We observed that FOXM1 was indeed activated by phosphorylation upon estrogen stimulation and silencing of FOXM1 attenuated the relevant gene signature. Analysis of scRNA-seq data from patient samples confirmed the existence of these divergent cell groups, with the FOXM1 signature predominantly found in ER negative cells. Further, multi-omic single-cell experiments indicated that the different cell groups have distinct chromatin accessibility states. Our results provide a comprehensive insight into ER biology at the single-cell level and potential therapeutic strategies to mitigate resistance to therapy.
Tandem DNA lesions, containing two contiguously damaged nucleotides, are commonly formed by ionizing radiation. Their effects on replication in mammalian cells are largely unknown. Replication of isolated 2-deoxyribonolactone (L) and thymine glycol (Tg), and the tandem lesion 5'-LTg was examined in human cells. Although nearly 100% of Tg was bypassed in HEK 293T cells, L was a significant replication block. 5'-LTg was an even stronger replication block with 5% TLS efficiency. Mutation frequency (MF) of Tg was 3.4%, which increased to 3.9% and 4.8% in pol ιand pol κ-deficient cells, respectively. An even greater increase in MF of Tg (to ~5.5%) was observed in cells with deficiency of both pol κ and pol ζ, suggesting that they work together to bypass Tg in an error-free manner. Isolated L bypass generated 12-18% one-base deletions, which increased as much as 60% in TLS polymerase-deficient cells. The fraction of deletion products also increased in TLS polymerase-deficient cells upon 5'-LTg bypass. In full-length products, dA was preferentially incorporated opposite an isolated L, as well as when it was part of a tandem lesion, in all cell types. However, misincorporation opposite Tg increased significantly when it was part of a tandem lesion. In wild type cells, targeted mutations increased about 3-fold to 9.7%, and to 17.4%, 15.9%, and 28.8%, respectively, in pol κ-, pol ζand pol ι-deficient cells. Overall, Tg is significantly more miscoding as part of a tandem lesion, and error-free Tg replication in HEK 293T cells requires participation of the TLS polymerases.
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