ssDNA Regions Research Articles

SLFN11 inhibits checkpoint maintenance and homologous recombination repair.

High expression levels of SLFN11 correlate with the sensitivity of human cancer cells to DNA-damaging agents. However, little is known about the underlying mechanism. Here, we show that SLFN11 interacts directly with RPA1 and is recruited to sites of DNA damage in an RPA1-dependent manner. Furthermore, we establish that SLFN11 inhibits checkpoint maintenance and homologous recombination repair by promoting the destabilization of the RPA-ssDNA complex, thereby sensitizing cancer cell lines expressing high endogenous levels of SLFN11 to DNA-damaging agents. Finally, we demonstrate that the RPA1-binding ability of SLFN11 is required for its function in the DNA damage response. Our findings not only provide novel insight into the molecular mechanisms underlying the drug sensitivity of cancer cell lines expressing SLFN11 at high levels, but also suggest that SLFN11 expression can serve as a biomarker to predict responses to DNA-damaging therapeutic agents.

Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting.

The metabolism of DNA in cells relies on the balance between hybridized double-stranded DNA (dsDNA) and local de-hybridized regions of ssDNA that provide access to binding proteins. Traditional melting experiments, in which short pieces of dsDNA are heated up until the point of melting into ssDNA, have determined that AT-rich sequences have a lower binding energy than GC-rich sequences. In cells, however, the double-stranded backbone of DNA is destabilized by negative supercoiling, and not by temperature. To investigate what the effect of GC content is on DNA melting induced by negative supercoiling, we studied DNA molecules with a GC content ranging from 38% to 77%, using single-molecule magnetic tweezer measurements in which the length of a single DNA molecule is measured as a function of applied stretching force and supercoiling density. At low force (<0.5pN), supercoiling results into twisting of the dsDNA backbone and loop formation (plectonemes), without inducing any DNA melting. This process was not influenced by the DNA sequence. When negative supercoiling is introduced at increasing force, local melting of DNA is introduced. We measured for the different DNA molecules a characteristic force F char, at which negative supercoiling induces local melting of the dsDNA. Surprisingly, GC-rich sequences melt at lower forces than AT-rich sequences: F char = 0.56pN for 77% GC but 0.73pN for 38% GC. An explanation for this counterintuitive effect is provided by the realization that supercoiling densities of a few percent only induce melting of a few percent of the base pairs. As a consequence, denaturation bubbles occur in local AT-rich regions and the sequence-dependent effect arises from an increased DNA bending/torsional energy associated with the plectonemes. This new insight indicates that an increased GC-content adjacent to AT-rich DNA regions will enhance local opening of the double-stranded DNA helix.

Melting of Duplex DNA in the Absence of ATP by the NS3 Helicase Domain through Specific Interaction with a Single-Strand/Double-Strand Junction

Helicases unwind double-stranded nucleic acids, remove secondary structures from single-stranded nucleic acids, and remove proteins bound to nucleic acids. For many helicases, the mechanisms for these different functions share the ability to translocate with a directional bias as a result of ATP binding and hydrolysis. Nonstructural protein 3 (NS3) is an essential enzyme expressed by the hepatitis C virus (HCV) and is known to catalyze the unwinding of both DNA and RNA substrates in a 3'-to-5' direction. We investigated the role of nucleic acid binding in the unwinding mechanism by examining ATP-independent unwinding. We observed that even in the absence of ATP, the NS3 helicase domain (NS3h) unwound duplexes only when they contained a 3'-tail (i.e., 3'-to-5' directionality). Blunt-ended duplexes and 5'-tailed duplexes were not melted even in the presence of a large excess concentration of the protein. NS3h was found to diffuse rapidly along single-stranded DNA at a rate of 30 nucleotides(2) s(-1). Upon encountering an appropriate single-strand/double-strand (ss/ds) junction, NS3h slowly melted the duplex under conditions with an excess protein concentration relative to DNA concentration. When a biotin-streptavidin block was placed into the ssDNA region, no melting of DNA was observed, suggesting that NS3h must diffuse along the ssDNA, and that the streptavidin blocked the diffusion. We conclude that the specific interaction between NS3h and the ss/dsDNA junction, coupled with diffusion, allows binding energy to melt duplex DNA with a directional bias. Alternatively, we found that the full-length NS3 protein did not exhibit strict directionality and was dependent on duplex DNA length. NS3 was able to unwind the duplex even in the presence of the biotin-streptavidin block. We propose a noncanonical model of unwinding for NS3 in which the enzyme binds directly to the duplex via protein-protein interactions to melt the substrate.

Interplay between Ku and Replication Protein A in the Restriction of Exo1-mediated DNA Break End Resection

DNA double-strand breaks can be eliminated via non-homologous end joining or homologous recombination. Non-homologous end joining is initiated by the association of Ku with DNA ends. In contrast, homologous recombination entails nucleolytic resection of the 5'-strands, forming 3'-ssDNA tails that become coated with replication protein A (RPA). Ku restricts end access by the resection nuclease Exo1. It is unclear how partial resection might affect Ku engagement and Exo1 restriction. Here, we addressed these questions in a reconstituted system with yeast proteins. With blunt-ended DNA, Ku protected against Exo1 in a manner that required its DNA end-binding activity. Despite binding poorly to ssDNA, Ku could nonetheless engage a 5'-recessed DNA end with a 40-nucleotide (nt) ssDNA overhang, where it localized to the ssDNA-dsDNA junction and efficiently blocked resection by Exo1. Interestingly, RPA could exclude Ku from a partially resected structure with a 22-nt ssDNA tail and thus restored processing by Exo1. However, at a 40-nt tail, Ku remained stably associated at the ssDNA-dsDNA junction, and RPA simultaneously engaged the ssDNA region. We discuss a model in which the dynamic equilibrium between Ku and RPA binding to a partially resected DNA end influences the timing and efficiency of the resection process.

Okazaki fragment maturation involves α-segment error editing by the mammalian FEN1/MutSα functional complex.

During nuclear DNA replication, proofreading-deficient DNA polymerase α (Pol α) initiates Okazaki fragment synthesis with lower fidelity than bulk replication by proofreading-proficient Pol δ or Pol ε. Here, we provide evidence that the exonuclease activity of mammalian flap endonuclease (FEN1) excises Pol α replication errors in a MutSα-dependent, MutLα-independent mismatch repair process we call Pol α-segment error editing (AEE). We show that MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5' ends of DNA substrates. Mouse cells and mice encoding FEN1 mutations display AEE deficiency, a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility. The results identify a novel role for FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism.

Improved artificial origins for phage Φ29 terminal protein-primed replication. Insights into early replication events.

The replication machinery of bacteriophage Φ29 is a paradigm for protein-primed replication and it holds great potential for applied purposes. To better understand the early replication events and to find improved origins for DNA amplification based on the Φ29 system, we have studied the end-structure of a double-stranded DNA replication origin. We have observed that the strength of the origin is determined by a combination of factors. The strongest origin (30-fold respect to wt) has the sequence CCC at the 3′ end of the template strand, AAA at the 5′ end of the non-template strand and 6 nucleotides as optimal unpairing at the end of the origin. We also show that the presence of a correctly positioned displaced strand is important because origins with 5′ or 3′ ssDNA regions have very low activity. Most of the effect of the improved origins takes place at the passage between the terminal protein-primed and the DNA-primed modes of replication by the DNA polymerase suggesting the existence of a thermodynamic barrier at that point. We suggest that the template and non-template strands of the origin and the TP/DNA polymerase complex form series of interactions that control the critical start of terminal protein-primed replication.

Diffusion of Human Replication Protein A along Single-Stranded DNA

Replication protein A (RPA) is a eukaryotic single-stranded DNA (ssDNA) binding protein that plays critical roles in most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA–DNA interactions, we combined ensemble and single-molecule fluorescence approaches to examine human RPA (hRPA) diffusion along ssDNA and find that an hRPA heterotrimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one-dimensional diffusion coefficient of hRPA on ssDNA of D1~5000nt2 s−1 at 37°C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA.

Binding affinity of ssDNA is improved by attachment of dsDNA regions

LidNA, a microRNA inhibitor consisting of a microRNA binding ssDNA region sandwiched between dsDNA regions had higher affinity to target oligonucleotides than that without dsDNA region. This enhancement in affinity was found to be owing to the suppressed mobility of ssDNA region by the presence of dsDNA regions.

Watching Aid Scanning Single Stranded and Transcribed DNA with Single Molecule Resolution

The activation-induced deoxycytidine deaminase (AID) is a member the Apobec family of enzymes that catalyzes dC to dU deamination on ssDNA tri-nucleotide motifs. In B cells, it is required to generate antibody diversity by initiating somatic hypermutation (SHM) in the variable region of immunoglobulin genes and class-switch recombination (CSR) in immunoglobulin switch regions. In turn, SHM and CSR are required to generate high-affinity antibodies that bind and neutralize invading antigens. Thus, AID plays an indispensable role in causing mutational diversity to enhance fitness and optimize the immune response.Here, we have used single-molecule fluorescence resonance energy transfer (smFRET) to visualize co-transcriptional scanning of AID. Our data show that AID can follow an active RNA polymerase directionally and processively with speeds upwards of 200 nt/s. However, transcription-stalling leads to bidirectional scanning in the transcription bubble, which in turn, provides AID the necessary time window to carry out deaminations. In bear ssDNA, AID scanning is slow (∼1 s-1), random and bi-directional. The enzyme remains bound to the ssDNA for ∼250 s on average. During this time, it can scan large (>70 nt) ssDNA regions, and it exhibits 'quasi-localization' near favorable deamination motifs. AID also creases the ssDNA during scanning in a sequence dependant manner.

Domain Architecture of RecQ Helicase Defines Mechanochemical Linkage via Multipartite Interactions with DNA Substrate during Unwinding Activity

RecQ helicases are ubiquitous enzymes that safeguard the genome by playing multiple roles in DNA repair, replication and recombination. They are unique in their capability to process a wide range of non-canonical structures associated with DNA metabolic intermediates. DNA-binding domains linked to the RecQ helicase core, including the winged-helix (WHD) and helicase-and-RnaseD-C-terminal (HRDC) domains, are thought to confer substrate specificity and modulate enzymatic activities. We combined ensemble biophysical and single-molecule magnetic tweezers assays to determine the mechanochemical linkage between ATP hydrolytic and DNA-restructuring activities of E. coli RecQ constructs of varying domain architecture. We compared the activities of wild-type, HRDC point mutant and deletion mutant, and WHD-HRDC deletion mutant constructs using several defined DNA structures and experimental geometries. We show that the WHD enhances unwinding processivity by stabilizing enzyme-DNA interactions, whereas the HRDC domain increases the overall DNA affinity but hinders the unwinding and ATPase activities of the helicase through interactions with single-stranded (ss) DNA regions. Intriguingly, HRDC-ssDNA interactions stabilize the pausing of the helicase during unwinding of a DNA hairpin in which both nascent ssDNA strands are mechanically strained, but not that of gapped duplex DNA in which one of the liberated ssDNA strands is mechanically unconstrained. Our study reveals that both the DNA substrate geometry and the contribution of auxiliary DNA-binding domains greatly influence the mechanochemical behavior of the helicase and the outcome of DNA-processing reactions, bearing consequences on the diverse in vivo actions of RecQ enzymes.

Nucleosome Positioning on Lambda DNA for Single-Molecule Analysis of Chromatin Remodeling - Development of a Versatile Lambda DNA Construct Capable of Reception of any DNA Sequences of Interest

Single-molecule biophysics and enzymology has become a vast field of investigation of biomolecules in the last decade. Application of these techniques enabled the characterization of molecular phenomena in a previously unprecedented resolution and highlighted previously hidden mechanisms of actions of enzymes. Lambda phage DNA is widely used for studying DNA biophysics and protein-DNA interactions because of its optimal size for single-molecule manipulations. However, single-molecule analysis of reactions that require specific DNA sequences or specialized DNA structures, including positioned nucleosomes to study chromatin remodeling, have been an unresolved problem. Here we demonstrate the development of a versatile lambda phage system (λKytos) modified to receive any types of DNA in the middle of lambda DNA. We integrated an array of nucleosome positioning sequences into λKytos and reconstituted fluorescently labeled nucleosomes on this substrate. Our results show that there is a strong bias for the nucleosomes to bind to the positioning sequences along the lambda DNA (30% of the nucleosomes bound to the positioning region which is 3% of the total length). The reconstituted chromatin can be visualized by optical trapping and TIRF microscopy that enables us to study sequence specific chromatin remodeling with a previously inaccessible resolution. We also demonstrate applications of the λKytos system where we introduced a replication origin, a single-stranded (ss) DNA gap, and a DNA bubble consisting of two non-pairing ssDNA region in the middle of λKytos. These substrates - and similar derivatives - will greatly facilitate single-molecule studies of DNA replication, recombination and repair in the presence and absence of reconstituted chromatin.

Direct Observation of RecBCD Helicase as Single-Stranded DNA Translocases

The heterotrimeric Escherichia coli RecBCD enzyme comprises two helicase motors with different polarities: RecB (3'-to-5') and RecD (5'-to-3'). This superfamily I helicase is responsible for initiating DNA double-strand-break (DSB) repair in the homologous recombination pathway. We used single-molecule tethered particle motion (TPM) experiments to visualize the RecBCD helicase translocation over long single-stranded (ss) DNA (>200 nt) with no apparent secondary structure. The bead-labeled RecBCD helicases were found to bind to the surface-immobilized blunt-end DNA, and translocate along the DNA substrates containing an ssDNA gap, resulting in a gradual decrease in the bead Brownian motion. Successful observation of RecBCD translocation over a long gap in either 3'-to-5' or 5'-to-3' ssDNA direction indicates that RecBCD helicase possesses ssDNA translocase activities in both polarities. Most RecBCD active tethers showed full translocation across the ssDNA to the dsDNA region, with about 19% of enzymes dissociated from the ss/dsDNA junction after translocating across the ssDNA region. In addition, we prepared DNA substrates containing two opposite polarities (5'-to-3' and 3'-to-5') of ssDNA regions intermitted by duplex DNA. RecBCD was able to translocate across both ssDNA regions in either ssDNA orientation orders, with less than 40% of tethers dissociating when entering into the second ssDNA region. These results suggest a model that RecBCD is able to switch between ssDNA translocases and rethread the other strand of ssDNA.

APOBEC3G cytosine deamination hotspots are defined by both sequence context and single-stranded DNA secondary structure

Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (i.e., APOBEC3G or A3G) is an evolutionarily conserved cytosine deaminase that potently restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons and other viruses. A3G has a nucleotide target site specificity for cytosine dinucleotides, though only certain cytosine dinucleotides are ‘hotspots’ for cytosine deamination, and others experience little or no editing by A3G. The factors that define these critical A3G hotspots are not fully understood. To investigate how A3G hotspots are defined, we used an in vitro fluorescence resonance energy transfer-based oligonucleotide assay to probe the site specificity of A3G. Our findings strongly suggest that the target single-stranded DNA (ssDNA) secondary structure as well as the bases directly 3′ and 5′ of the cytosine dinucleotide are critically important A3G recognition. For instance, A3G cannot readily deaminate a cytosine dinucleotide in ssDNA stem structures or in nucleotide base loops composed of three bases. Single-stranded nucleotide loops up to seven bases in length were poor targets for A3G activity unless cytosine residues flanked the cytosine dinucleotide. Furthermore, we observed that A3G favors adenines, cytosines and thymines flanking the cytosine dinucleotide target in unstructured regions of ssDNA. Low cytosine deaminase activity was detected when guanines flanked the cytosine dinucleotide. Taken together, our findings provide the first demonstration that A3G cytosine deamination hotspots are defined by both the sequence context of the cytosine dinucleotide target as well as the ssDNA secondary structure. This knowledge can be used to better trace the origins of mutations to A3G activity, and illuminate its impact on processes such as HIV-1 genetic variation.

FANC Pathway Promotes UV-Induced Stalled Replication Forks Recovery by Acting Both Upstream and Downstream Polη and Rev1

To cope with ultraviolet C (UVC)-stalled replication forks and restart DNA synthesis, cells either undergo DNA translesion synthesis (TLS) by specialised DNA polymerases or tolerate the lesions using homologous recombination (HR)-based mechanisms. To gain insight into how cells manage UVC-induced stalled replication forks, we analysed the molecular crosstalk between the TLS DNA polymerases Polη and Rev1, the double-strand break repair (DSB)-associated protein MDC1 and the FANC pathway. We describe three novel functional interactions that occur in response to UVC-induced DNA lesions. First, Polη and Rev1, whose optimal expression and/or relocalisation depend on the FANC core complex, act upstream of FANCD2 and are required for the proper relocalisation of monoubiquitinylated FANCD2 (Ub-FANCD2) to subnuclear foci. Second, during S-phase, Ub-FANCD2 and MDC1 relocalise to UVC-damaged nuclear areas or foci simultaneously but independently of each other. Third, Ub-FANCD2 and MDC1 are independently required for optimal BRCA1 relocalisation. While RPA32 phosphorylation (p-RPA32) and RPA foci formation were reduced in parallel with increasing levels of H2AX phosphorylation and MDC1 foci in UVC-irradiated FANC pathway-depleted cells, MDC1 depletion was associated with increased UVC-induced Ub-FANCD2 and FANCD2 foci as well as p-RPA32 levels and p-RPA32 foci. On the basis of the previous observations, we propose that the FANC pathway participates in the rescue of UVC-stalled replication forks in association with TLS by maintaining the integrity of ssDNA regions and by preserving genome stability and preventing the formation of DSBs, the resolution of which would require the intervention of MDC1.

Direct Observation of Recbcd Helicase as ssDNA Translocases

The E. coli RecBCD is a heterotrimeric enzyme composed of two helicase motors with different polarities: RecB (3’-to-5’) and RecD (5’-to-3’). This Superfamily I helicase is responsible for the initiation of DNA double-strand-break (DSB) repair in the homologous recombination pathway. We used single-molecule tethered particle motion (TPM) experiments to visualize the RecBCD helicase translocation over long single-stranded (ss) DNA (> 200 nt) without secondary structure. The bead-labeled RecBCD helicases were found to bind to the surface-immobilized blunt duplex DNA, and translocate along the duplex/single-stranded/duplex DNA substrate, resulting in a gradual decrease in the bead Brownian motion. Successful observation of RecBCD translocation over long ssDNA gap in either 3’-to-5’ or 5’-to-3’ direction indicates that both RecB and RecD are ssDNA translocases. We also applied continuous force (∼ 0.2 pN) to stretch DNA substrates and to remove any potentially transient looped ssDNA structure, and observed continuous translocation of RecBCD. It confirms the ssDNA translocase activities of RecBCD helicase. About 78 % of active tethers showed full translocation across the ssDNA to the dsDNA region, and the other 22 % enzymes dissociated from the ss/dsDNA junction after translocating across the ssDNA region. We also prepared a double-gapped substrate containing two regions of ssDNA with opposite polarities (5’-to-3’ and 3’-to-5’) intermitted by duplex DNA. RecBCD was able to translocate across both ssDNA regions in either ssDNA orientation orders, with 20 - 40 % of tethers dissociating while entering the second ssDNA region. These results suggest a mechanism that RecBCD is able to switch motors and rethread into the other strand after translocating along an ssDNA gapped region.

A Binary Molecular Gate Made of DNA and Protein

Many proteins have evolved the ability to recognize and bind to specific sequences of DNA. This specificity is quite high and often is the basis for genetic switches which can repress or induce transcription in cells. One of the best known DNA-binding proteins is the lac repressor protein which binds tightly to the lac operator but loses its affinity for this site upon the binding of lactose. We have used this lac repressor protein and the lac operator sequence to construct a binary molecular gate which can be controlled by the effector molecule lactose. The device is constructed of three DNA strands which form an elongated hairpin structure of two dsDNA arms with a short ssDNA hinge at one end, and two ssDNA regions on the opposite end. A fourth ssDNA molecule (the 'locking' strand), complementary to the two ssDNA regions, binds by complementary base pairing to seal the gate in the closed configuration. Addition of lac repressor protein to the device causes a displacement of the locking strand by binding to two lac operators embedded in the double-stranded hairpin structure. Binding of the proteins is rapid and sufficiently strong to cause a displacement of the locking strand, switching the gate into the open conformation. Addition of lactose quickly causes a release of the lac repressor protein from the operators allowing the gate to be closed by the binding of the locking strand. The opening and closing of this binary gate was detected by gel shift assays and by fluorescence resonance energy transfer spectroscopy of two dyes located near the opening of this gate. The gate could be cycled repeatedly, responded uniquely to lactose, and may be useful as a device for moving or holding structures on a molecular scale.

Base Damage within Single-Strand DNA Underlies In Vivo Hypermutability Induced by a Ubiquitous Environmental Agent

Chromosomal DNA must be in single-strand form for important transactions such as replication, transcription, and recombination to occur. The single-strand DNA (ssDNA) is more prone to damage than double-strand DNA (dsDNA), due to greater exposure of chemically reactive moieties in the nitrogenous bases. Thus, there can be agents that damage regions of ssDNA in vivo while being inert toward dsDNA. To assess the potential hazard posed by such agents, we devised an ssDNA–specific mutagenesis reporter system in budding yeast. The reporter strains bear the cdc13-1 temperature-sensitive mutation, such that shifting to 37°C results in telomere uncapping and ensuing 5′ to 3′ enzymatic resection. This exposes the reporter region, containing three closely-spaced reporter genes, as a long 3′ ssDNA overhang. We validated the ability of the system to detect mutagenic damage within ssDNA by expressing a modified human single-strand specific cytosine deaminase, APOBEC3G. APOBEC3G induced a high density of substitutions at cytosines in the ssDNA overhang strand, resulting in frequent, simultaneous inactivation of two reporter genes. We then examined the mutagenicity of sulfites, a class of reactive sulfur oxides to which humans are exposed frequently via respiration and food intake. Sulfites, at a concentration similar to that found in some foods, induced a high density of mutations, almost always as substitutions at cytosines in the ssDNA overhang strand, resulting in simultaneous inactivation of at least two reporter genes. Furthermore, sulfites formed a long-lived adducted 2′-deoxyuracil intermediate in DNA that was resistant to excision by uracil–DNA N-glycosylase. This intermediate was bypassed by error-prone translesion DNA synthesis, frequently involving Pol ζ, during repair synthesis. Our results suggest that sulfite-induced lesions in DNA can be particularly deleterious, since cells might not possess the means to repair or bypass such lesions accurately.

A high‐throughput, flow cytometry‐based method to quantify DNA‐end resection in mammalian cells

Replication protein A (RPA) is an essential trimeric protein complex that binds to single-stranded DNA (ssDNA) in eukaryotic cells and is involved in various aspects of cellular DNA metabolism, including replication and repair. Although RPA is ubiquitously expressed throughout the cell cycle, it localizes to DNA replication forks during S phase, and is recruited to sites of DNA damage when regions of ssDNA are exposed. During DNA double-strand break (DSB) repair by homologous recombination (HR), RPA recruitment to DNA damage sites depends on a process termed DNA-end resection. Consequently, RPA recruitment to sub-nuclear regions bearing DSBs has been used as readout for resection and for ongoing HR. Quantification of RPA recruitment by immunofluorescence-based microscopy techniques is time consuming and requires extensive image analysis of relatively small populations of cells. Here, we present a high-throughput flow-cytometry method that allows the use of RPA staining to measure cell proliferation and DNA-damage repair by HR in an unprecedented, unbiased and quantitative manner. © 2012 International Society for Advancement of Cytometry

Mutational showers during carcinogenesis

Mutational showers during carcinogenesis

Human Embryonic Stem Cells Fail to Activate CHK1 and Commit to Apoptosis in Response to DNA Replication Stress

Pluripotent cells of the early embryo, to which embryonic stem cells (ESCs) correspond, give rise to all the somatic cells of the developing fetus. Any defects that occur in their genome or epigenome would have devastating consequences. Genetic and epigenetic change in human ESCs appear to be an inevitable consequence of long-term culture, driven by selection of variant cells that have a higher propensity for self-renewal rather than either differentiation or death. Mechanisms underlying the potentially separate events of mutation and subsequent selection of variants are poorly understood. Here, we show that human ESCs and their malignant counterpart, embryonal carcinoma (EC) cells, both fail to activate critical S-phase checkpoints when exposed to DNA replication inhibitors and commit to apoptosis instead. Human ESCs and EC cells also fail to form replication protein A, γH2AX, or RAD51 foci or load topoisomerase (DNA) II binding protein 1 onto chromatin in response to replication inhibitors. Furthermore, direct measurements of single-stranded DNA (ssDNA) show that these cells fail to generate the ssDNA regions in response to replication stress that are necessary for the activation of checkpoints and the initiation of homologous recombination repair to protect replication fork integrity and restart DNA replication. Taken together, our data suggest that pluripotent cells control genome integrity by the elimination of damaged cells through apoptosis rather than DNA repair, and therefore, mutations or epigenetic modifications resulting in an imbalance in cell death control could lead to genetic instability.