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

Abstract

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.

Enzymatic bypass of an N6-deoxyadenosine DNA–ethylene dibromide–peptide cross-link by translesion DNA polymerases

“…TLS polymerases have been shown to bypass a GSH-based adduct ( 52 , 69 , 70 ) and (N-terminal-linked) peptides as long as 5- and 12-mers ( 71 , 72 , 73 ) but not a 23-mer peptide or green fluorescent protein ( 72 ). In vitro studies, including some in cells, have shown the mutagenic bypass of DNA–peptide cross-link by TLS polymerases ( 11 , 42 , 74 ). It is important to know how a DNA polymerase can bypass each cross-linked peptide entity and potentially cause base-pair mutations.…”

“…Mammalian TLS polymerases include Y-family Pol η, ⍳, and κ and Rev1 ( 41 ), which have low processivity and fidelity ( 39 ). Only in a few cases has the mutagenic potential of a DNA–peptide cross-link been analyzed ( 42 ), and essentially all of the work in this area has been done with either uncharacterized cross-links or synthetic models.…”

DNA polymerases η and κ bypass N2-guanine-O6-alkylguanine DNA alkyltransferase cross-linked DNA-peptides

“…Early studies from the Lloyd laboratory showed that bacterial DNA polymerases could efficiently bypass 4-mer- and 12-mer peptides linked to DNA by a ring-opened γ-hydroxypropano dG adduct ( 47 , 48 ). Since then, several studies have been done with human TLS polymerases that can bypass DNA–peptide cross-links ( 19 , 49 , 50 ), although the number of reports is relatively small. However, these have involved shorter peptides (10-mers), and in one study a 23-mer posed a strong block to replication by hpol η, ι, and κ ( 50 ).…”

DNA polymerase ι compensates for Fanconi anemia pathway deficiency by countering DNA replication stress

“…Indeed, Pol ι has been shown to be able to bypass bulky adducts that are unable to be bypassed by other translesion synthesis (TLS) polymerases (74). Furthermore, in the presence of Mn 2+ , Pol ι is able to bypass a peptide adduct that mimics the DNA-protein crosslink repair intermediate (75,76). This ability may constitute an effective alternative in countering the accumulation of crosslinking damage from FA pathway loss.…”