Replication and repair of a reduced 2΄-deoxyguanosine-abasic site interstrand cross-link in human cells

Abstract

Apurinic/apyrimidinic (AP) sites, or abasic sites, which are a common type of endogenous DNA damage, can forge interstrand DNA–DNA cross-links via reaction with the exocyclic amino group on a nearby 2΄-deoxyguanosine or 2΄-deoxyadenosine in the opposite strand. Here, we utilized a shuttle vector method to examine the efficiency and fidelity with which a reduced dG–AP cross-link-containing plasmid was replicated in cultured human cells. Our results showed that the cross-link constituted strong impediments to DNA replication in HEK293T cells, with the bypass efficiencies for the dG- and AP-containing strands being 40% and 20%, respectively. While depletion of polymerase (Pol) η did not perturb the bypass efficiency of the lesion, the bypass efficiency was markedly reduced (to 1–10%) in the isogenic cells deficient in Pol κ, Pol ι or Pol ζ, suggesting the mutual involvement of multiple translesion synthesis polymerases in bypassing the lesion. Additionally, replication of the cross-linked AP residue in HEK293T cells was moderately error-prone, inducing a total of ∼26% single-nucleobase substitutions at the lesion site, whereas replication past the cross-linked dG component occurred at a mutation frequency of ∼8%. Together, our results provided important insights into the effects of an AP-derived interstrand cross-link on the efficiency and accuracy of DNA replication in human cells.

Remodeling of Interstrand Crosslink Proximal Replisomes Is Dependent on ATR, FANCM, and FANCD2

“…Among the most potent blocks to replication are interstrand crosslinks (ICLs). They are formed by highly toxic cancer chemotherapy drugs and DNA reactive compounds generated during normal cellular metabolism, including lipid peroxidation products (Niedernhofer et al, 2003), aldehydes (Pontel et al, 2015), and abasic sites (Price et al, 2017). ICLs are of additional interest because of the hyper-sensitivity to them of cells from patients with Fanconi anemia (FA).…”

Structure of a Stable Interstrand DNA Cross-Link Involving a β-N-Glycosyl Linkage Between an N6-dA Amino Group and an Abasic Site

“…Studies using the bacteriophage ϕ29 DNA polymerase demonstrated that if not repaired, these ICLs block replication . The reduced dG-AP ICL required multiple polymerases for bypass and was mutagenic …”

Mitochondrial transcription factor A promotes DNA strand cleavage at abasic sites

“…Importantly, AP sites are chemically labile and reactive and can lead to secondary DNA damage such as DNA single-strand breaks (SSBs), DNA-DNA interstrand cross-links, and DNAprotein cross-links (DPCs) (18)(19)(20)(21)(22)(23). If not repaired, AP sites and their derivatives are mutagenic in the nuclear genome (24)(25)(26); however, the understanding of the biological consequence of AP sites in mitochondria remains limited.…”