Translesion polymerase eta both facilitates DNA replication and promotes increased human genetic variation at common fragile sites

Abstract

Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. They are hotspots for chromosomal instability in cancer. Repetitive sequences located at CFS loci are inefficiently copied by replicative DNA polymerase (Pol) delta. However, translesion synthesis Pol eta has been shown to efficiently polymerize CFS-associated repetitive sequences in vitro and facilitate CFS stability by a mechanism that is not fully understood. Here, by locus-specific, single-molecule replication analysis, we identified a crucial role for Pol eta (encoded by the gene POLH) in the in vivo replication of CFSs, even without exogenous stress. We find that Pol eta deficiency induces replication pausing, increases initiation events, and alters the direction of replication-fork progression at CFS-FRA16D in both lymphoblasts and fibroblasts. Furthermore, certain replication pause sites at CFS-FRA16D were associated with the presence of non-B DNA-forming motifs, implying that non-B DNA structures could increase replication hindrance in the absence of Pol eta. Further, in Pol eta-deficient fibroblasts, there was an increase in fork pausing at fibroblast-specific CFSs. Importantly, while not all pause sites were associated with non-B DNA structures, they were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. From these findings, we propose that Pol eta replicating through CFSs may result in genetic variations found in the human population at these sites.

Function and Molecular Mechanism of the DNA Damage Response in Immunity and Cancer Immunotherapy

“…During SHM, AID deaminates a particular trinucleotide sequence in ssDNA of transcriptionally active genes, leaving behind numerous uracil residues and producing predominantly nucleotide substitutions in rearranged V genes on the heavy- and light-chain loci, and switch (S) regions, which precede most C genes on the heavy chain locus ( 217 , 218 ). The mutagenic outcome of uracil lesions can then be determined by one of the following DDR responses: (i) Uracil can act as a template for replication, resulting in a fixed C-T transition mutation; (ii) U-G mismatches can be recognized by the error‐prone MMR machinery, in which the MutSα complex (MSH2-MSH6) detects the mismatch and recruits MutLα complex (MLH1-PMS2) to nick the DNA, followed by the recruitment of Polη (DNA polymerase η) to generate mutations ( 219 , 220 ); (iii) Non‐canonical BER initiated by uracil DNA glycosylase (UNG) can be used to recruit proliferating cell nuclear antigen (PCNA) at the lesions, and low‐fidelity polymerases such as Polη, which can increase mutations during replication of common DNA fragile sites ( 221 ), then can be recruited by PCNA ubiquitination and utilized by both MMR and BER resulting in mutagenic repair ( 149 , 222 – 224 ). The nick generated by the UNG-dependent BER pathway is particularly important for CSR, as UNG1 knock out largely abolishes CSR ( 225 ).…”

Global landscape of replicative DNA polymerase usage in the human genome

“…While noncanonical polymerase usage has mainly been associated with DNA damage tolerance, there is increasing evidence for such phenomena under unperturbed conditions, particularly in mammalian cells [37][38][39][40][41] . Our data thus raise the important question of how extensively the unanticipated level of the plasticity of DNA polymerase usage underpins replication of the human genome.…”

Polymerase η Recruits DHX9 Helicase to Promote Replication across Guanine Quadruplex Structures

“…DNA polymerase η (Pol η) displays accurate TLS over UV-induced DNA lesions, especially cyclobutane pyrimidine dimers. − Mutations in the POLH gene, which encodes Pol η, result in the variant form of xeroderma pigmentosum (XPV) syndrome in humans. , Pol η also assumes important functions in several other related processes, including bypassing a broad spectrum of other DNA lesions and unusual DNA structures, such as fragile sites and guanine quadruplexes (G4s) …”

“…Furthermore, genetic depletion of Pol η renders U2OS cells sensitive to telomestatin, a G4-binding ligand . Cellular assays also revealed the function of Pol η in promoting replication across common fragile sites, and genetic ablation of Pol η in mice led to senescence in adipose tissues …”

“…4−6 Mutations in the POLH gene, which encodes Pol η, result in the variant form of xeroderma pigmentosum (XPV) syndrome in humans. 5,6 Pol η also assumes important functions in several other related processes, including bypassing a broad spectrum of other DNA lesions 7 and unusual DNA structures, such as fragile sites 8 and guanine quadruplexes (G4s). 9 We reason that a comprehensive assessment about Pol ηinteracting proteins at the global proteome scale may offer new insights into its functions.…”