5-Formylcytosine mediated DNA-peptide cross-link induces predominantly semi-targeted mutations in both Escherichia coli and human cells.

Histone proteins can become trapped on DNA in the presence of 5-formylcytosine (5fC) to form toxic DNA-protein conjugates. Their repair may involve proteolytic digestion resulting in DNA-peptide cross-links (DpCs). Here, we have investigated replication of a model DpC comprised of an 11-mer peptide (NH2-GGGKGLGK∗GGA) containing an oxy-lysine residue (K∗) conjugated to 5fC in DNA. Both CXG and CXT (where X = 5fC-DpC) sequence contexts were examined. Replication of both constructs gave low viability (<10%) in Escherichia coli, whereas TLS efficiency was high (72%) in HEK 293T cells. In E. coli, the DpC was bypassed largely error-free, inducing only 2 to 3% mutations, which increased to 4 to 5% with SOS. For both sequences, semi-targeted mutations were dominant, and for CXG, the predominant mutations were G→T and G→C at the 3'-base to the 5fC-DpC. In HEK 293T cells, 7 to 9% mutations occurred, and the dominant mutations were the semi-targeted G → T for CXG and T → G for CXT. These mutations were reduced drastically in cells deficient in hPol η, hPol ι or hPol ζ, suggesting a role of these TLS polymerases in mutagenic TLS. Steady-state kinetics studies using hPol η confirmed that this polymerase induces G → T and T → G transversions at the base immediately 3' to the DpC. This study reveals a unique replication pattern of 5fC-conjugated DpCs, which are bypassed largely error-free in both E. coli and human cells and induce mostly semi-targeted mutations at the 3' position to the lesion.

Site-Specific 5-Formyl Cytosine Mediated DNA-Histone Cross-Links: Synthesis and Polymerase Bypass by Human DNA Polymerase η.

DNA-protein cross-links (DPCs) between DNA epigenetic mark 5-formylC and lysine residues of histone proteins spontaneously form in human cells. Such conjugates are likely to influence chromatin structure and mediate DNA replication, transcription, and repair, but are challenging to study due to their reversible nature. Here we report the construction of site specific, hydrolytically stable DPCs between 5fdC in DNA and K4 of histone H3 and an investigation of their effects on DNA replication. Our approach employs oxime ligation, allowing for site-specific conjugation of histones to DNA under physiological conditions. Primer extension experiments revealed that histone H3-DNA crosslinks blocked DNA synthesis by hPol η polymerase, but were bypassed following proteolytic processing.

Translesion Synthesis Past 5-Formylcytosine-Mediated DNA-Peptide Cross-Links by hPolη Is Dependent on the Local DNA Sequence.

DNA-protein cross-links (DPCs) are unusually bulky DNA lesions that form when cellular proteins become trapped on DNA following exposure to ultraviolet light, free radicals, aldehydes, and transition metals. DPCs can also form endogenously when naturally occurring epigenetic marks [5-formyl cytosine (5fC)] in DNA react with lysine and arginine residues of histones to form Schiff base conjugates. Our previous studies revealed that DPCs inhibit DNA replication and transcription but can undergo proteolytic cleavage to produce smaller DNA-peptide conjugates. We have shown that 5fC-conjugated DNA-peptide cross-links (DpCs) placed within the CXA sequence (X = DpC) can be bypassed by human translesion synthesis (TLS) polymerases η and κ in an error-prone manner. However, the local nucleotide sequence context can have a strong effect on replication bypass of bulky lesions by influencing the geometry of the ternary complex among the DNA template, polymerase, and the incoming dNTP. In this work, we investigated polymerase bypass of 5fC-DNA-11-mer peptide cross-links placed in seven different sequence contexts (CXC, CXG, CXT, CXA, AXA, GXA, and TXA) in the presence of human TLS polymerase η. Primer extension products were analyzed by gel electrophoresis, and steady-state kinetics of the misincorporation of dAMP opposite the DpC lesion in different base sequence contexts was investigated. Our results revealed a strong impact of nearest neighbor base identity on polymerase η activity in the absence and presence of a DpC lesion. Molecular dynamics simulations were used to structurally explain the experimental findings. Our results suggest a possible role of local DNA sequence in promoting TLS-related mutational hot spots in the presence and absence of DpC lesions.

Transcriptional Bypass of DNA-Protein and DNA-Peptide Conjugates by T7 RNA Polymerase.

DNA-protein cross-links (DPCs) are unusually bulky DNA adducts that block the access of proteins to DNA and interfere with gene expression, replication, and repair. We previously described DPC formation at the N7-guanine position of DNA in human cells treated with antitumor nitrogen mustards and platinum compounds and have shown that DPCs can form endogenously at DNA epigenetic mark 5-formyl-dC. However, insufficient information is available about the effects of these structurally distinct DPCs on transcription. In the present work, we employ a combination of in vitro assays, mass spectrometry, and molecular dynamics simulations to examine the ability of phage T7 RNA polymerase to bypass DPCs conjugated to the C7 position of 7-deaza-dG and the C5 position of dC. These model adducts represent endogenous DPCs induced by exposure to antitumor drugs and formed at epigenetics DNA marks, respectively. Our results reveal that DPCs containing full-length proteins significantly inhibit in vitro transcription by T7 RNA polymerase, while short DNA-peptide cross-links (DpCs) are bypassed. DpCs conjugated to the C7 position of 7-deaza-dG are transcribed with high fidelity, while the same polypeptides attached to the C5 position of dC induce transcription errors. Molecular dynamics simulations of DpCs conjugated either to the C5 atom of dC or the C7 position of 7-deaza-dG on the template strand in T7 RNA polymerase explain how the conjugated peptide can be accommodated in the narrow major groove of the DNA-RNA hybrid and how the modified dC can form a stable mismatch with the incoming ATP in the polymerase active site, allowing for transcriptional mutagenesis.

Error-prone replication of a 5-formylcytosine-mediated DNA-peptide cross-link in human cells.

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 site-specific 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 C→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 hPol ι or hPol ζ reduced the mutation frequency by nearly 50%. However, the most significant reduction in mutation frequency (50%-70%) was observed upon simultaneous knockout of hPol η and hPol κ with knockdown of hPol ζ, 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 δ and hPol ϵ 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.