DNA and Chromosomes
Extended DNA-binding interfaces beyond the canonical SAP domain contribute to the function of replication stress regulator SDE2 at DNA replication forks
Elevated DNA replication stress causes instability of the DNA replication fork and increased DNA mutations, which underlies tumorigenesis. The DNA replication stress regulator silencing-defective 2 (SDE2) is known to bind to TIMELESS (TIM), a protein of the fork protection complex, and enhances its stability, thereby supporting replisome activity at DNA replication forks. However, the DNA-binding activity of SDE2 is not well defined. Here, we structurally and functionally characterize a new conserved DNA-binding motif related to the SAP (SAF-A/B, Acinus, PIAS) domain in human SDE2 and establish its preference for ssDNA.Stable G-quadruplex DNA structures promote replication-dependent genome instability
G-quadruplex (G4)–prone structures are abundant in mammalian genomes, where they have been shown to influence DNA replication, transcription, and genome stability. In this article, we constructed cells with a single ectopic homopurine/homopyrimidine repeat tract derived from the polycystic kidney disease type 1 (PKD1) locus, which is capable of forming triplex (H3) and G4 DNA structures. We show that ligand stabilization of these G4 structures results in deletions of the G4 consensus sequence, as well as kilobase deletions spanning the G4 and ectopic sites.Lamin A/C recruits ssDNA protective proteins RPA and RAD51 to stalled replication forks to maintain fork stability
Lamin A/C provides a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. Lamin A/C dysfunction is associated with cancer, aging, and degenerative diseases. The mechanisms whereby lamin A/C regulates genome stability remain poorly understood. We demonstrate a crucial role for lamin A/C in DNA replication. Lamin A/C binds to nascent DNA, especially during replication stress (RS), ensuring the recruitment of replication fork protective factors RPA and RAD51.Interdomain connecting loop and J loop structures determine cross-species compatibility of PCNA
Eukaryotic proliferating cell nuclear antigen (PCNA) plays an essential role in orchestrating the assembly of the replisome complex, stimulating processive DNA synthesis, and recruiting other regulatory proteins during the DNA damage response. PCNA and its binding partner network are relatively conserved in eukaryotes, and it exhibits extraordinary structural similarity across species. However, despite this structural similarity, the PCNA of a given species is rarely functional in heterologous systems.Enzymatic bypass of an N6-deoxyadenosine DNA–ethylene dibromide–peptide cross-link by translesion DNA polymerases
Unrepaired DNA–protein cross-links, due to their bulky nature, can stall replication forks and result in genome instability. Large DNA–protein cross-links can be cleaved into DNA–peptide cross-links, but the extent to which these smaller fragments disrupt normal replication is not clear. Ethylene dibromide (1,2-dibromoethane) is a known carcinogen that can cross-link the repair protein O6-alkylguanine-DNA alkyltransferase (AGT) to the N6 position of deoxyadenosine (dA) in DNA, as well as four other positions in DNA.Age and insulin-like growth factor-1 impact PCNA monoubiquitination in UVB-irradiated human skin
Nonmelanoma skin cancers occur primarily in individuals over the age of 60 and are characterized by an abundance of ultraviolet (UV) signature mutations in keratinocyte DNA. Though geriatric skin removes UV photoproducts from DNA less efficiently than young adult skin, it is not known whether the utilization of other prosurvival but potentially mutagenic DNA damage tolerance systems such as translesion synthesis (TLS) is altered in older individuals. Using monoubiquitination of the replicative DNA polymerase clamp protein PCNA (proliferating cell nuclear antigen) as a biochemical marker of TLS pathway activation, we find that UVB exposure of the skin of individuals over the age of 65 results in a higher level of PCNA monoubiquitination than in the skin of young adults.Enzymatic bypass and the structural basis of miscoding opposite the DNA adduct 1,N2-ethenodeoxyguanosine by human DNA translesion polymerase η
Etheno (ε)-adducts, e.g., 1,N2-ε−guanine (1,N2-ε-G) and 1,N6-ε−adenine (1,N6-ε-A), are formed through the reaction of DNA with metabolites of vinyl compounds or with lipid peroxidation products. These lesions are known to be mutagenic, but it is unknown how they lead to errors in DNA replication that are bypassed by DNA polymerases. Here we report the structural basis of misincorporation frequencies across from 1,N2-ε-G by human DNA polymerase (hpol) η. In single-nucleotide insertions opposite the adduct 1,N2-ε-G, hpol η preferentially inserted dGTP, followed by dATP, dTTP, and dCTP.Ada protein– and sequence context–dependent mutagenesis of alkyl phosphotriester lesions in Escherichia coli cells
Alkyl phosphotriester (alkyl-PTE) lesions are frequently induced in DNA and are resistant to repair. Here, we synthesized and characterized methyl (Me)- and n-butyl (nBu)-PTEs in two diastereomeric configurations (Sp and Rp) at six different flanking dinucleotide sites, i.e. XT and TX (X = A, C, or G), and assessed how these lesions impact DNA replication in Escherichia coli cells. When single-stranded vectors contained an Sp-Me-PTE in the sequence contexts of 5′-AT-3′, 5′-CT-3′, or 5′-GT-3′, DNA replication was highly efficient and the replication products for all three sequence contexts contained 85–90% AT and 5–10% TG.Genetic evidence for reconfiguration of DNA polymerase θ active site for error-free translesion synthesis in human cells
The action mechanisms revealed by the biochemical and structural analyses of replicative and translesion synthesis (TLS) DNA polymerases (Pols) are retained in their cellular roles. In this regard, DNA polymerase θ differs from other Pols in that whereas purified Polθ misincorporates an A opposite 1,N6-ethenodeoxyadenosine (ϵdA) using an abasic-like mode, Polθ performs predominantly error-free TLS in human cells. To test the hypothesis that Polθ adopts a different mechanism for replicating through ϵdA in human cells than in the purified Pol, here we analyze the effects of mutations in the two highly conserved tyrosine residues, Tyr-2387 and Tyr-2391, in the Polθ active site.The roles of polymerases ν and θ in replicative bypass of O6- and N2-alkyl-2′-deoxyguanosine lesions in human cells
Exogenous and endogenous chemicals can react with DNA to produce DNA lesions that may block DNA replication. Not much is known about the roles of polymerase (Pol) ν and Pol θ in translesion synthesis (TLS) in cells. Here we examined the functions of these two polymerases in bypassing major-groove O6-alkyl-2′-deoxyguanosine (O6-alkyl-dG) and minor-groove N2-alkyl-dG lesions in human cells, where the alkyl groups are ethyl, n-butyl (nBu), and, for O6-alkyl-dG, pyridyloxobutyl. We found that Pol ν and Pol θ promote TLS across major-groove O6-alkyl-dG lesions.Molecular and structural characterization of oxidized ribonucleotide insertion into DNA by human DNA polymerase β
During oxidative stress, inflammation, or environmental exposure, ribo- and deoxyribonucleotides are oxidatively modified. 8-Oxo-7,8-dihydro-2′-guanosine (8-oxo-G) is a common oxidized nucleobase whose deoxyribonucleotide form, 8-oxo-dGTP, has been widely studied and demonstrated to be a mutagenic substrate for DNA polymerases. Guanine ribonucleotides are analogously oxidized to r8-oxo-GTP, which can constitute up to 5% of the rGTP pool. Because ribonucleotides are commonly misinserted into DNA, and 8-oxo-G causes replication errors, we were motivated to investigate how the oxidized ribonucleotide is utilized by DNA polymerases.Repair and translesion synthesis of O6-alkylguanine DNA lesions in human cells
O6-alkyl-2′-deoxyguanosine (O6-alkyl-dG) lesions are among the most mutagenic and prevalent alkylated DNA lesions that are associated with cancer initiation and progression. In this study, using a shuttle vector–based strand-specific PCR-competitive replication and adduct bypass assay in conjunction with tandem MS for product identification, we systematically assessed the repair and replicative bypass of a series of O6-alkyl-dG lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, in several human cell lines.The abundant DNA adduct N7-methyl deoxyguanosine contributes to miscoding during replication by human DNA polymerase η
Aside from abasic sites and ribonucleotides, the DNA adduct N7-methyl deoxyguanosine (N7-CH3 dG) is one of the most abundant lesions in mammalian DNA. Because N7-CH3 dG is unstable, leading to deglycosylation and ring-opening, its miscoding potential is not well-understood. Here, we employed a 2′-fluoro isostere approach to synthesize an oligonucleotide containing an analog of this lesion (N7-CH3 2′-F dG) and examined its miscoding potential with four Y-family translesion synthesis DNA polymerases (pols): human pol (hpol) η, hpol κ, and hpol ι and Dpo4 from the archaeal thermophile Sulfolobus solfataricus.Human DNA polymerase η has reverse transcriptase activity in cellular environments
Classical DNA and RNA polymerase (pol) enzymes have defined roles with their respective substrates, but several pols have been found to have multiple functions. We reported previously that purified human DNA pol η (hpol η) can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) and can use both DNA and RNA as substrates. X-ray crystal structures revealed that two pol η residues, Phe-18 and Tyr-92, behave as steric gates to influence sugar selectivity.DNA replication studies of N-nitroso compound–induced O6-alkyl-2′-deoxyguanosine lesions in Escherichia coli
N-Nitroso compounds (NOCs) are common DNA-alkylating agents, are abundantly present in food and tobacco, and can also be generated endogenously. Metabolic activation of some NOCs can give rise to carboxymethylation and pyridyloxobutylation/pyridylhydroxybutylation of DNA, which are known to be carcinogenic and can lead to gastrointestinal and lung cancer, respectively. Herein, using the competitive replication and adduct bypass (CRAB) assay, along with MS- and NMR-based approaches, we assessed the cytotoxic and mutagenic properties of three O6-alkyl-2′-deoxyguanosine (O6-alkyl-dG) adducts, i.e.Replication protein A dynamically regulates monoubiquitination of proliferating cell nuclear antigen
DNA damage tolerance permits bypass of DNA lesions encountered during S-phase and may be carried out by translesion DNA synthesis (TLS). Human TLS requires selective monoubiquitination of proliferating cell nuclear antigen (PCNA) sliding clamps encircling damaged DNA. This posttranslational modification (PTM) is catalyzed by Rad6/Rad18. Recent studies revealed that replication protein A (RPA), the major ssDNA-binding protein, is involved in the regulation of PCNA monoubiquitination and interacts directly with Rad18 on chromatin and in the nucleoplasm.Cytotoxic and mutagenic properties of O6-alkyl-2′-deoxyguanosine lesions in Escherichia coli cells
Environmental exposure and cellular metabolism can give rise to DNA alkylation, which can occur on the nitrogen and oxygen atoms of nucleobases, as well as on the phosphate backbone. Although O6-alkyl-2′-deoxyguanosine (O6-alkyl-dG) lesions are known to be associated with cancer, not much is known about how the alkyl group structures in these lesions affect their repair and replicative bypass in vivo or how translesion synthesis DNA polymerases influence the latter process. To answer these questions, here we synthesized oligodeoxyribonucleotides harboring seven O6-alkyl-dG lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, or sBu, and examined the impact of these lesions on DNA replication in Escherichia coli cells.Cytotoxic and mutagenic properties of minor-groove O2-alkylthymidine lesions in human cells
Endogenous metabolism, environmental exposure, and cancer chemotherapy can lead to alkylation of DNA. It has been well documented that, among the different DNA alkylation products, minor-groove O2-alkylthymidine (O2-alkyldT) lesions are inefficiently repaired. In the present study, we examined how seven O2-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, are recognized by the DNA replication machinery in human cells. We found that the replication bypass efficiencies of these lesions decrease with increasing length of the alkyl chain, and that these lesions induce substantial frequencies of T→A and T→G mutations.Impact of tobacco-specific nitrosamine–derived DNA adducts on the efficiency and fidelity of DNA replication in human cells
The tobacco-derived nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) are known human carcinogens. Following metabolic activation, NNK and NNN can induce a number of DNA lesions, including several 4-(3-pyridyl)-4-oxobut-1-yl (POB) adducts. However, it remains unclear to what extent these lesions affect the efficiency and accuracy of DNA replication and how their replicative bypass is influenced by translesion synthesis (TLS) DNA polymerases. In this study, we investigated the effects of three stable POB DNA adducts (O2-POB-dT, O4-POB-dT, and O6-POB-dG) on the efficiency and fidelity of DNA replication in HEK293T human cells.Polymerase θ-helicase efficiently unwinds DNA and RNA-DNA hybrids
POLQ is a unique multifunctional replication and repair gene that encodes for a N-terminal superfamily 2 helicase and a C-terminal A-family polymerase. Although the function of the polymerase domain has been investigated, little is understood regarding the helicase domain. Multiple studies have reported that polymerase θ-helicase (Polθ-helicase) is unable to unwind DNA. However, it exhibits ATPase activity that is stimulated by single-stranded DNA, which presents a biochemical conundrum. In contrast to previous reports, we demonstrate that Polθ-helicase (residues 1–894) efficiently unwinds DNA with 3′–5′ polarity, including DNA with 3′ or 5′ overhangs, blunt-ended DNA, and replication forks.Genetic control of predominantly error-free replication through an acrolein-derived minor-groove DNA adduct
Acrolein, an α,β-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from metabolic oxidation of polyamines, and it is a ubiquitous environmental pollutant. The reaction of acrolein with the N2 of guanine in DNA leads to the formation of γ-hydroxy-1-N2-propano-2′ deoxyguanosine (γ-HOPdG), which can exist in DNA in a ring-closed or a ring-opened form. Here, we identified the translesion synthesis (TLS) DNA polymerases (Pols) that conduct replication through the permanently ring-opened reduced form of γ-HOPdG ((r) γ-HOPdG) and show that replication through this adduct is mediated via Rev1/Polη-, Polι/Polκ-, and Polθ-dependent pathways, respectively.Replisome-mediated translesion synthesis by a cellular replicase
Genome integrity relies on the ability of the replisome to navigate ubiquitous DNA damage during DNA replication. The Escherichia coli replisome transiently stalls at leading-strand template lesions and can either reinitiate replication downstream of the lesion or recruit specialized DNA polymerases that can bypass the lesion via translesion synthesis. Previous results had suggested that the E. coli replicase might play a role in lesion bypass, but this possibility has not been tested in reconstituted DNA replication systems.Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea
Incorporation of ribonucleotides during DNA replication has severe consequences for genome stability. Although eukaryotes possess a number of redundancies for initiating and completing repair of misincorporated ribonucleotides, archaea such as Thermococcus rely only upon RNaseH2 to initiate the pathway. Because Thermococcus DNA polymerases incorporate as many as 1,000 ribonucleotides per genome, RNaseH2 must be efficient at recognizing and nicking at embedded ribonucleotides to ensure genome integrity.Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks
Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5′ end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5′-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks.Metnase Mediates Loading of Exonuclease 1 onto Single Strand Overhang DNA for End Resection at Stalled Replication Forks
Stalling at DNA replication forks generates stretches of single-stranded (ss) DNA on both strands that are exposed to nucleolytic degradation, potentially compromising genome stability. One enzyme crucial for DNA replication fork repair and restart of stalled forks in human is Metnase (also known as SETMAR), a chimeric fusion protein consisting of a su(var)3–9, enhancer-of-zeste and trithorax (SET) histone methylase and transposase nuclease domain. We previously showed that Metnase possesses a unique fork cleavage activity necessary for its function in replication restart and that its SET domain is essential for recovery from hydroxyurea-induced DNA damage.
