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Common fragile sites (CFSs) are inherently unstable genomic loci that are recurrently altered in human tumor cells. Despite their instability, CFS are ubiquitous throughout the human genome and associated with large tumor suppressor genes or oncogenes. CFSs are enriched with repetitive DNA sequences, one feature postulated to explain why these loci are inherently difficult to replicate, and sensitive to replication stress. We have shown that specialized DNA polymerases (Pols) η and κ replicate CFS-derived sequences more efficiently than the replicative Pol δ. However, we lacked an understanding of how these enzymes cooperate to ensure efficient CFS replication. Here, we designed a model of lagging strand replication with RFC loaded PCNA that allows for maximal activity of the four-subunit human Pol δ holoenzyme, Pol η, and Pol κ in polymerase mixing assays. We discovered that Pol η and κ are both able to exchange with Pol δ stalled at repetitive CFS sequences, enhancing Normalized Replication Efficiency. We used this model to test the impact of PCNA mono-ubiquitination on polymerase exchange, and found no change in polymerase cooperativity in CFS replication compared with unmodified PCNA. Finally, we modeled replication stress in vitro using aphidicolin and found that Pol δ holoenzyme synthesis was significantly inhibited in a dose-dependent manner, preventing any replication past the CFS. Importantly, Pol η and κ were still proficient in rescuing this stalled Pol δ synthesis, which may explain, in part, the CFS instability phenotype of aphidicolin-treated Pol η and Pol κ-deficient cells. In total, our data support a model wherein Pol δ stalling at CFSs allows for free exchange with a specialized polymerase that is not driven by PCNA. [ABSTRACT FROM AUTHOR]

G-quadruplexes (G4s), a type of non-B DNA, play important roles in a wide range of molecular processes, including replication, transcription, and translation. Genome integrity relies on efficient and accurate DNA synthesis, and is compromised by various stressors, to which non-B DNA structures such as G4s can be particularly vulnerable. However, the impact of G4 structures on DNA polymerase fidelity is largely unknown. Using an in vitro forward mutation assay, we investigated the fidelity of human DNA polymerases delta (δ4, four-subunit), eta (η), and kappa (κ) during synthesis of G4 motifs representing those in the human genome. The motifs differ in sequence, topology, and stability, features that may affect DNA polymerase errors. Polymerase error rate hierarchy (δ4 < κ < η) is largely maintained during G4 synthesis. Importantly, we observed unique polymerase error signatures during synthesis of VEGF G4 motifs, stable G4s which form parallel topologies. These statistically significant errors occurred within, immediately flanking, and encompassing the G4 motif. For pol δ4, the errors were deletions, insertions and complex errors within the G4 or encompassing the G4 motif and surrounding sequence. For pol η, the errors occurred in 3' sequences flanking the G4 motif. For pol κ, the errors were frameshift mutations within G-tracts of the G4. Because these error signatures were not observed during synthesis of an antiparallel G4 and, to a lesser extent, a hybrid G4, we suggest that G4 topology and/or stability could influence polymerase fidelity. Using in silico analyses, we show that most polymerase errors are predicted to have minimal effects on predicted G4 stability. Our results provide a unique view of G4s not previously elucidated, showing that G4 motif heterogeneity differentially influences polymerase fidelity within the motif and flanking sequences. Thus, our study advances the understanding of how DNA polymerase errors contribute to G4 mutagenesis. • G-quadruplex (G4) mutagenesis is dependent on G4 motif sequence and DNA polymerase. • The G4 motif influences polymerase errors in the 3' flanking sequence. • Large-scale polymerase errors are dependent on G4 topology with parallel strands. • Most polymerase errors do not substantially impact predicted G4 stability. • Polymerases δ, κ, and η can create errors that completely abolish G4 formation. [ABSTRACT FROM AUTHOR]

Highlights: [•] Human cell lines that express a variant form of Pol κ (F171A) were established. [•] The cells exhibited lower mutation frequency toward benzo[a]pyrene DNA adducts. [•] Phenylalanine 171 is a molecular brake for the translesion DNA synthesis by Pol κ. [•] Pol κ is not well tuned to bypass benzo[a]pyrene DNA adducts. [•] The cognate substances for Pol κ may be lesions induced by endogenous mutagens. [Copyright &y& Elsevier]

Abstract: To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous sources of DNA damage. DNA integrity is maintained by the coordinated action of DNA damage response mechanisms and DNA repair. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage but are potentially error-prone. Here, we investigate the role of DNA polymerase κ (pol κ) in TLS across alkylated lesions by silencing this polymerase (pol) in human cells using transient small RNA interference. We show that human pol κ has a significant protective role against methyl nitrosourea (MNU)-associated cytotoxicity without affecting significantly mutagenicity. The increase in MNU-induced cytotoxicity when pol κ is down-regulated was affected by the levels of O6-methylguanine DNA methyltransferase and fully abolished when mismatch repair (MMR) was defective. Following MNU treatment, the cell cycle profile was unaffected by the pol κ status. The downregulation of pol κ caused a severe delay in the onset of the second mitosis that was fully dependent on the presence of O6-methylguanine (O6-meGua) lesions. After MNU exposure, in the absence of pol κ, the frequency of sister chromatid exchanges was unaffected whereas the induction of RAD 51 foci increased. We propose that pol κ partially protects human cells from the MMR-dependent cytotoxicity of O6-meGua lesions by restoring the integrity of replicated duplexes containing single-stranded gaps generated opposite O6-meGua facilitated by RAD 51 binding. [Copyright &y& Elsevier]

Abstract: Human cells possess multiple specialized DNA polymerases (Pols) that bypass a variety of DNA lesions which otherwise would block chromosome replication. Human polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N 2-deoxyguanine (BPDE-N 2 -dG) DNA adducts in an almost error-free manner. To better understand the relationship between the structural features in the active site and lesion bypass by Pol κ, we mutated codons corresponding to amino acids appearing close to the adducts in the active site, and compared bypass efficiencies. Remarkably, the substitution of alanine for phenylalanine 171 (F171), an amino acid conserved between Pol κ and its bacterial counterpart Escherichia coli DinB, enhanced the efficiencies of dCMP incorporation opposite (−)- and (+)-trans-anti-BPDE-N 2-dG 18-fold. This substitution affected neither the fidelity of TLS nor the efficiency of dCMP incorporation opposite normal guanine. This amino acid change also enhanced the binding affinity of Pol κ to template/primer DNA containing (−)-trans-anti-BPDE-N 2-dG. These results suggest that F171 functions as a molecular brake for TLS across BPDE-N 2-dG by Pol κ and that the F171A derivative of Pol κ bypasses these DNA lesions more actively than does the wild-type enzyme. [Copyright &y& Elsevier]

Abstract: The efficiency and fidelity of nucleotide incorporation and next-base extension by DNA polymerase (pol) κ past N 2-ethyl-Gua were measured using steady-state and rapid kinetic analyses. DNA pol κ incorporated nucleotides and extended 3′ termini opposite N 2-ethyl-Gua with measured efficiencies and fidelities similar to that opposite Gua indicating a role for DNA pol κ at the insertion and extension steps of N 2-ethyl-Gua bypass. The DNA pol κ was maximally activated to similar levels by a twenty-fold lower concentration of Mn2+ compared to Mg2+. In addition, the steady state analysis indicated that high fidelity DNA pol κ-catalyzed N 2-ethyl-Gua bypass is Mg2+-dependent. Strikingly, Mn2+ activation of DNA pol κ resulted in a dramatically lower efficiency of correct nucleotide incorporation opposite both N 2-ethyl-Gua and Gua compared to that detected upon Mg2+ activation. This effect is largely governed by diminished correct nucleotide binding as indicated by the high Km values for dCTP insertion opposite N 2-ethyl-Gua and Gua with Mn2+ activation. A rapid kinetic analysis showed diminished burst amplitudes in the presence of Mn2+ compared to Mg2+ indicating that DNA pol κ preferentially utilizes Mg2+ activation. These kinetic data support a DNA pol κ wobble base pairing mechanism for dCTP incorporation opposite N 2-ethyl-Gua. Furthermore, the dramatically different polymerization efficiencies of the Y-family DNA pols κ and ι in the presence of Mn2+ suggest a metal ion-dependent regulation in coordinating the activities of these DNA pols during translesion synthesis. [ABSTRACT FROM AUTHOR]

Abstract: Talaroflavone (1) and 1-deoxyrubralactone (2) are natural compounds isolated from cultures of a fungal strain derived from sea algae, and their structures were determined by spectroscopic analyses. Compound 2 is a novel rubralactone derivative, 6-hydroxy-8-methoxy-1-methyl-1,2,3a,9b-tetrahydrocyclopenta[c]isochromene-3,5-dione. These compounds selectively inhibited the activities of families X and Y of eukaryotic DNA polymerases (pols), and compound 2 was a stronger inhibitor than compound 1. The IC50 values of compound 2 on rat pol β, which is a pol of family X, and human pol κ, which is a pol of family Y, were 11.9 and 59.8μM, respectively. On the other hand, compounds 1 and 2 did not influence the activities of the other families of eukaryotic pols, such as family A (i.e., pol γ) and family B (i.e., pols α, δ, and ε), and showed no effect even on the activities of plant pols α and β, prokaryotic pols, and other DNA metabolic enzymes, such as calf primase of pol α, human immunodeficiency virus type-1 (HIV-1) reverse transcriptase, human telomerase, T7 RNA polymerase, mouse IMP dehydrogenase (type II), human topoisomerase I and II, T4 polynucleotide kinase, and bovine deoxyribonuclease I. This is the first report about the selective inhibitors of families X and Y of eukaryotic pols. [Copyright &y& Elsevier]

During the immune response to T cell-dependent antigen, somatic hypermutation (SHM) is introduced into immunoglobulin (Ig) genes. The variable region is the target for SHM and it is here that DNA lesions are introduced and mutations are generated. It has been suggested that error-prone DNA polymerase(s) may play an important role in this mutagenesis phase. Recently, DNA polymerase κ (Polκ), which belongs to the Y-family of DNA polymerases, was identified. Since a hot spot of SHMs (RGYW motif) is also a hot spot of mutations by human Polκ, this enzyme was suggested to be an SHM instigator. In order to address the question whether Polκ is involved in SHM, we immunized Polκ-deficient mice and analyzed the SHM of the Ig heavy chain gene. We found that the SHM frequency and spectrum were indistinguishable between the Polκ knockout mice and control mice. These results suggested that Polκ is not essential for this process. [Copyright &y& Elsevier]