Stepwise requirements for polymerases δ and θ in theta-mediated end joining.

Timely repair of chromosomal double-strand breaks is required for genome integrity and cellular viability. The polymerase theta-mediated end joining pathway has an important role in resolving these breaks and is essential in cancers defective in other DNA repair pathways, thus making it an emerging therapeutic target1. It requires annealing of 2-6 nucleotides of complementary sequence, microhomologies, that are adjacent to the broken ends, followed by initiation of end-bridging DNA synthesis by polymerase θ. However, the other pathway steps remain inadequately defined, and the enzymes required for them are unknown. Here we demonstrate requirements for exonucleolytic digestion of unpaired 3' tails before polymerase θ can initiate synthesis, then a switch to a more accurate, processive and strand-displacing polymerase to complete repair. We show the replicative polymerase, polymerase δ, is required for both steps; its 3' to 5' exonuclease activity for flap trimming, then its polymerase activity for extension and completion of repair. The enzymatic steps that are essential and specific to this pathway are mediated by two separate, sequential engagements of the two polymerases. The requisite coupling of these steps together is likely to be facilitated by physical association of the two polymerases. This pairing of polymerase δ with a polymerase capable of end-bridging synthesis, polymerase θ, may help to explain why the normally high-fidelity polymerase δ participates in genome destabilizing processes such as mitotic DNA synthesis2 and microhomology-mediated break-induced replication3.

A basal-level activity of ATR links replication fork surveillance and stress response.

Mammalian cells use diverse pathways to prevent deleterious consequences during DNA replication, yet the mechanism by which cells survey individual replisomes to detect spontaneous replication impediments at the basal level, and their accumulation during replication stress, remain undefined. Here, we used single-molecule localization microscopy coupled with high-order-correlation image-mining algorithms to quantify the composition of individual replisomes in single cells during unperturbed replication and under replicative stress. We identified a basal-level activity of ATR that monitors and regulates the amounts of RPA at forks during normal replication. Replication-stress amplifies the basal activity through the increased volume of ATR-RPA interaction and diffusion-driven enrichment of ATR at forks. This localized crowding of ATR enhances its collision probability, stimulating the activation of its replication-stress response. Finally, we provide a computational model describing how the basal activity of ATR is amplified to produce its canonical replication stress response.

Innate immunity gene Nod2 protects mice from orthotopic breast cancer.

BACKGROUND: Nod2 is involved in innate immune responses to bacteria, regulation of metabolism, and sensitivity to cancer. A Nod2 polymorphism is associated with breast cancer, but the role of Nod2 in the development and progression of breast cancer is unknown. METHODS: Here, we tested the hypothesis that Nod2 protects mice from breast cancer using the 4T1 orthotopic model of mammary tumorigenesis. WT and Nod2-/- mice were injected with 4T1 mammary carcinoma cells and the development of tumors was monitored. A detailed analysis of the tumor transcriptome was performed and genes that were differentially expressed and pathways that were predicted to be altered between WT and Nod2-/- mice were identified. The activation of key signaling molecules involved in metabolism and development of cancer was studied. RESULTS: Our data demonstrate that Nod2-/- mice had a higher incidence and larger tumors than WT mice. Nod2-/- mice had increased expression of genes that promote DNA replication and cell division, and decreased expression of genes required for lipolysis, lipogenesis, and steroid biosynthesis compared with WT mice. Nod2-/- mice also had lower expression of genes required for adipogenesis and reduced levels of lipids compared with WT mice. The tumors in Nod2-/- mice had decreased expression of genes associated with PPARα/γ signaling, increased activation of STAT3, decreased activation of STAT5, and no change in the activation of ERK compared with WT mice. CONCLUSIONS: We conclude that Nod2 protects mice from the 4T1 orthotopic breast tumor, and that tumors in Nod2-/- mice are predicted to have increased DNA replication and cell proliferation and decreased lipid metabolism compared with WT mice.

The Pglyrp1-Regulated Microbiome Enhances Experimental Allergic Asthma.

Changes in intestinal or respiratory microbiomes in infants correlate with increased incidence of asthma, but the causative role of microbiome in the susceptibility to asthma and the host genes that regulate these changes in microbiome are mostly unknown. In this study, we show that decreased responsiveness to allergic asthma in Pglyrp1 -/- mice (lacking bactericidal peptidoglycan recognition protein 1) could be transferred to germ-free wild-type mice by colonization of mothers and newborns with microbiota from Pglyrp1 -/- mice. These colonized mice had decreased airway resistance and fewer inflammatory cells, less severe histopathology, and lower levels of IgE and proallergic cytokines and chemokines in the lungs. This microbiome-dependent decreased responsiveness to asthma was most pronounced in colonized germ-free BALB/c mice (genetically predisposed to asthma), only partially evident in outbred germ-free Swiss Webster mice, and marginal in conventional BALB/c mice following depletion of microbiome with antibiotics. Mice with a low asthmatic response colonized with microbiota from Pglyrp1 -/- mice had increased abundance of Bacteroidetes and decreased abundance of Firmicutes, Tenericutes, Deferribacteres, and Spirochaetes in the feces and increased abundance of Pasteurella in the oropharynx. These changes in bacterial abundance in the feces and oropharynx correlated with lower asthmatic responses in the lungs. Thus, our results show that Pglyrp1 enhances allergic asthmatic responses primarily through its effect on the host intestinal microbiome and identify several bacteria that may increase or decrease sensitivity to asthma. This effect of microbiome is strong in asthma-prone BALB/c mice and weak in asthma-resistant outbred mice and requires germ-free conditions before colonization with microbiota from Pglyrp1 -/- mice.

ATR-Chk1 activation mitigates replication stress caused by mismatch repair-dependent processing of DNA damage.

The mismatch repair pathway (MMR) is essential for removing DNA polymerase errors, thereby maintaining genomic stability. Loss of MMR function increases mutation frequency and is associated with tumorigenesis. However, how MMR is executed at active DNA replication forks is unclear. This has important implications for understanding how MMR repairs O6-methylguanine/thymidine (MeG/T) mismatches created upon exposure to DNA alkylating agents. If MeG/T lesion recognition by MMR initiates mismatch excision, the reinsertion of a mismatched thymidine during resynthesis could initiate futile repair cycles. One consequence of futile repair cycles might be a disruption of overall DNA replication in the affected cell. Herein, we show that in MMR-proficient HeLa cancer cells, treatment with a DNA alkylating agent slows S phase progression, yet cells still progress into the next cell cycle. In the first S phase following treatment, they activate ataxia telangiectasia and Rad3-related (ATR)-Checkpoint Kinase 1 (Chk1) signaling, which limits DNA damage, while inhibition of ATR kinase activity accelerates DNA damage accumulation and sensitivity to the DNA alkylating agent. We also observed that exposure of human embryonic stem cells to alkylation damage severely compromised DNA replication in a MMR-dependent manner. These cells fail to activate the ATR-Chk1 signaling axis, which may limit their ability to handle replication stress. Accordingly, they accumulate double-strand breaks and undergo immediate apoptosis. Our findings implicate the MMR-directed response to alkylation damage as a replication stress inducer, suggesting that repeated MMR processing of mismatches may occur that can disrupt S phase progression.

Engineered Tug-of-War Between Kinesin and Dynein Controls Direction of Microtubule Based Transport In Vivo.

Bidirectional transport of membrane organelles along microtubules (MTs) is driven by plus-end directed kinesins and minus-end directed dynein bound to the same cargo. Activities of opposing MT motors produce bidirectional movement of membrane organelles and cytoplasmic particles along MT transport tracks. Directionality of MT-based transport might be controlled by a protein complex that determines which motor type is active at any given moment of time, or determined by the outcome of a tug-of-war between MT motors dragging cargo organelles in opposite directions. However, evidence in support of each mechanisms of regulation is based mostly on the results of theoretical analyses or indirect experimental data. Here, we test whether the direction of movement of membrane organelles in vivo can be controlled by the tug-of-war between opposing MT motors alone, by attaching a large number of kinesin-1 motors to organelles transported by dynein to minus-ends of MTs. We find that recruitment of kinesin significantly reduces the length and velocity of minus-end-directed dynein-dependent MT runs, leading to a reversal of the overall direction of dynein-driven organelles in vivo. Therefore, in the absence of external regulators tug-of-war between opposing MT motors alone is sufficient to determine the directionality of MT transport in vivo.

Bactericidal peptidoglycan recognition protein induces oxidative stress in Escherichia coli through a block in respiratory chain and increase in central carbon catabolism.

Mammalian Peptidoglycan Recognition Proteins (PGRPs) kill both Gram-positive and Gram-negative bacteria through simultaneous induction of oxidative, thiol and metal stress responses in bacteria. However, metabolic pathways through which PGRPs induce these bactericidal stress responses are unknown. We screened Keio collection of Escherichia coli deletion mutants and revealed that deleting genes for respiratory chain flavoproteins or for tricarboxylic acid (TCA) cycle resulted in increased resistance of E. coli to PGRP killing. PGRP-induced killing depended on the production of hydrogen peroxide, which required increased supply of NADH for respiratory chain oxidoreductases from central carbon catabolism (glycolysis and TCA cycle), and was controlled by cAMP-Crp. Bactericidal PGRP induced a rapid decrease in respiration, which suggested that the main source of increased production of hydrogen peroxide was a block in respiratory chain and diversion of electrons from NADH oxidoreductases to oxygen. CpxRA two-component system was a negative regulator of PGRP-induced oxidative stress. By contrast, PGRP-induced thiol stress (depletion of thiols) and metal stress (increase in intracellular free Zn2+ through influx of extracellular Zn2+ ) were mostly independent of oxidative stress. Thus, manipulating pathways that induce oxidative, thiol and metal stress in bacteria could be a useful strategy to design new approaches to antibacterial therapy.

How innate immunity proteins kill bacteria and why they are not prone to resistance.

Recent advances on antibacterial activity of peptidoglycan recognition proteins (PGRPs) offer some insight into how innate immunity has retained its antimicrobial effectiveness for millions of years with no frequent emergence of resistant strains. First, PGRP can bind to multiple components of bacterial envelope (peptidoglycan, lipoteichoic acid, and lipopolysaccharide). Second, PGRP simultaneously induces oxidative, thiol, and metal stress responses in bacteria, which individually are bacteriostatic, but in combination are bactericidal. Third, PGRP induces oxidative, thiol, and metal stress responses in bacteria through three independent pathways. Fourth, antibacterial effects of PGRP are enhanced by other innate immune responses. Thus, emergence of PGRP resistance is prevented by bacteriostatic effect and independence of each PGRP-induced stress response, as PGRP resistance would require simultaneous acquisition of three separate mechanisms disabling the induction of all three stress responses. By contrast, each antibiotic has one primary target and one primary antibacterial mechanism, and for this reason resistance to antibiotics can be generated by inhibition of this primary mechanism. Manipulating bacterial metabolic responses can enhance bacterial killing by antibiotics and elimination of antibiotic-tolerant bacteria, but such manipulations do not overcome genetically encoded antibiotic resistance. Pathogens cause infections by evading, inhibiting, or subverting host immune responses.

Peptidoglycan recognition proteins kill bacteria by inducing oxidative, thiol, and metal stress.

Mammalian Peptidoglycan Recognition Proteins (PGRPs) are a family of evolutionary conserved bactericidal innate immunity proteins, but the mechanism through which they kill bacteria is unclear. We previously proposed that PGRPs are bactericidal due to induction of reactive oxygen species (ROS), a mechanism of killing that was also postulated, and later refuted, for several bactericidal antibiotics. Here, using whole genome expression arrays, qRT-PCR, and biochemical tests we show that in both Escherichia coli and Bacillus subtilis PGRPs induce a transcriptomic signature characteristic of oxidative stress, as well as correlated biochemical changes. However, induction of ROS was required, but not sufficient for PGRP killing. PGRPs also induced depletion of intracellular thiols and increased cytosolic concentrations of zinc and copper, as evidenced by transcriptome changes and supported by direct measurements. Depletion of thiols and elevated concentrations of metals were also required, but by themselves not sufficient, for bacterial killing. Chemical treatment studies demonstrated that efficient bacterial killing can be recapitulated only by the simultaneous addition of agents leading to production of ROS, depletion of thiols, and elevation of intracellular metal concentrations. These results identify a novel mechanism of bacterial killing by innate immunity proteins, which depends on synergistic effect of oxidative, thiol, and metal stress and differs from bacterial killing by antibiotics. These results offer potential targets for developing new antibacterial agents that would kill antibiotic-resistant bacteria.

Peptidoglycan recognition protein 3 and Nod2 synergistically protect mice from dextran sodium sulfate-induced colitis.

Aberrant immune response and changes in the gut microflora are the main causes of inflammatory bowel disease (IBD). Peptidoglycan recognition proteins (Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4) are bactericidal innate immunity proteins that maintain normal gut microbiome, protect against experimental colitis, and are associated with IBD in humans. Nucleotide-binding oligomerization domain 2 (Nod2) is an intracellular bacterial sensor and may be required for maintaining normal gut microbiome. Mutations in Nod2 are strongly associated with Crohn's disease, but the causative mechanism is not understood, and the role of Nod2 in ulcerative colitis is not known. Because IBD is likely caused by variable multiple mutations in different individuals, in this study, we examined the combined role of Pglyrp3 and Nod2 in the development of experimental colitis in mice. We demonstrate that a combined deficiency of Pglyrp3 and Nod2 results in higher sensitivity to dextran sodium sulfate-induced colitis compared with a single deficiency. Pglyrp3(-/-)Nod2(-/-) mice had decreased survival and higher loss of body weight, increased intestinal bleeding, higher apoptosis of colonic mucosa, elevated expression of cytokines and chemokines, altered gut microbiome, and increased levels of ATP in the colon. Increased sensitivity to dextran sodium sulfate-induced colitis in Pglyrp3(-/-)Nod2(-/-) mice depended on increased apoptosis of intestinal epithelium, changed gut microflora, and elevated ATP. Pglyrp3 deficiency contributed colitis-predisposing intestinal microflora and increased intestinal ATP, whereas Nod2 deficiency contributed higher apoptosis and responsiveness to increased level of ATP. In summary, Pglyrp3 and Nod2 are both required for maintaining gut homeostasis and protection against colitis, but their protective mechanisms differ.

Human pluripotent stem cells have a novel mismatch repair-dependent damage response.

Human pluripotent stem cells (PSCs) are presumed to have robust DNA repair pathways to ensure genome stability. PSCs likely need to protect against mutations that would otherwise be propagated throughout all tissues of the developing embryo. How these cells respond to genotoxic stress has only recently begun to be investigated. Although PSCs appear to respond to certain forms of damage more efficiently than somatic cells, some DNA damage response pathways such as the replication stress response may be lacking. Not all DNA repair pathways, including the DNA mismatch repair (MMR) pathway, have been well characterized in PSCs to date. MMR maintains genomic stability by repairing DNA polymerase errors. MMR is also involved in the induction of cell cycle arrest and apoptosis in response to certain exogenous DNA-damaging agents. Here, we examined MMR function in PSCs. We have demonstrated that PSCs contain a robust MMR pathway and are highly sensitive to DNA alkylation damage in an MMR-dependent manner. Interestingly, the nature of this alkylation response differs from that previously reported in somatic cell types. In somatic cells, a permanent G2/M cell cycle arrest is induced in the second cell cycle after DNA damage. The PSCs, however, directly undergo apoptosis in the first cell cycle. This response reveals that PSCs rely on apoptotic cell death as an important defense to avoid mutation accumulation. Our results also suggest an alternative molecular mechanism by which the MMR pathway can induce a response to DNA damage that may have implications for tumorigenesis.

Peptidoglycan recognition protein 1 enhances experimental asthma by promoting Th2 and Th17 and limiting regulatory T cell and plasmacytoid dendritic cell responses.

Asthma is a common inflammatory disease involving cross-talk between innate and adaptive immunity. We reveal that antibacterial innate immunity protein, peptidoglycan recognition protein (Pglyrp)1, is involved in the development of allergic asthma. Pglyrp1(-/-) mice developed less severe asthma than wild-type (WT) mice following sensitization with house dust mite (allergen) (HDM). HDM-sensitized Pglyrp1(-/-) mice, compared with WT mice, had diminished bronchial hyperresponsiveness (lung airway resistance); numbers of eosinophils, neutrophils, lymphocytes, and macrophages in bronchoalveolar lavage fluid and lungs; inflammatory cell infiltrates in the lungs around bronchi, bronchioles, and pulmonary arteries and veins; lung remodeling (mucin-producing goblet cell hyperplasia and metaplasia and smooth muscle hypertrophy and fibrosis); levels of IgE, eotaxins, IL-4, IL-5, and IL-17 in the lungs; and numbers of Th2 and Th17 cells and expression of their marker genes in the lungs. The mechanism underlying this decreased sensitivity of Pglyrp1(-/-) mice to asthma was increased generation and activation of CD8α(+)ß(+) and CD8α(+)ß(-) plasmacytoid dendritic cells (pDC) and increased recruitment and activity of regulatory T (Treg) cells in the lungs. In vivo depletion of pDC in HDM-sensitized Pglyrp1(-/-) mice reversed the low responsive asthma phenotype of Pglyrp1(-/-) mice to resemble the more severe WT phenotype. Thus, Pglyrp1(-/-) mice efficiently control allergic asthma by upregulating pDC and Treg cells in the lungs, whereas in WT mice, Pglyrp1 is proinflammatory and decreases pDC and Treg cells and increases proasthmatic Th2 and Th17 responses. Blocking Pglyrp1 or enhancing pDC in the lungs may be beneficial for prevention and treatment of asthma.

Inactive Parp2 causes Tp53-dependent lethal anemia by blocking replication-associated nick ligation in erythroblasts.

PARP1&2 enzymatic inhibitors (PARPi) are promising cancer treatments. But recently, their use has been hindered by unexplained severe anemia and treatment-related leukemia. In addition to enzymatic inhibition, PARPi also trap PARP1&2 at DNA lesions. Here, we report that unlike Parp2 -/- mice, which develop normally, mice expressing catalytically-inactive Parp2 (E534A, Parp2 EA/EA ) succumb to Tp53- and Chk2 -dependent erythropoietic failure in utero , mirroring Lig1 -/- mice. While DNA damage mainly activates PARP1, we demonstrate that DNA replication activates PARP2 robustly. PARP2 is selectively recruited and activated by 5'-phosphorylated nicks (5'p-nicks) between Okazaki fragments, typically resolved by Lig1. Inactive PARP2, but not its active form or absence, impedes Lig1- and Lig3-mediated ligation, causing dose-dependent replication fork collapse, particularly harmful to erythroblasts with ultra-fast forks. This PARylation-dependent structural function of PARP2 at 5'p-nicks explains the detrimental effects of PARP2 inhibition on erythropoiesis, revealing the mechanism behind the PARPi-induced anemia and leukemia, especially those with TP53/CHK2 loss. Significance: This work shows that the hematological toxicities associated with PARP inhibitors stem not from impaired PARP1 or PARP2 enzymatic activity but rather from the presence of inactive PARP2 protein. Mechanistically, these toxicities reflect a unique role of PARP2 at 5'-phosphorylated DNA nicks during DNA replication in erythroblasts.

Dormant origin firing promotes head-on transcription-replication conflicts at transcription termination sites in response to BRCA2 deficiency.

BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome.

Peptidoglycan recognition protein Pglyrp2 protects mice from psoriasis-like skin inflammation by promoting regulatory T cells and limiting Th17 responses.

Skin protects the body from the environment and is an important component of the innate and adaptive immune systems. Psoriasis is a frequent inflammatory skin disease of unknown cause determined by multigenic predisposition, environmental factors, and aberrant immune response. Peptidoglycan recognition proteins (Pglyrps) are expressed in the skin, and we report in this article that they modulate sensitivity in an experimentally induced mouse model of psoriasis. We demonstrate that Pglyrp2(-/-) mice (but not Pglyrp3(-/-) and Pglyrp4(-/-) mice) are more sensitive to the development of 12-O-tetradecanoylphorbol 13-acetate-induced psoriasis-like inflammation, whereas Pglyrp1(-/-) mice are less sensitive. The mechanism underlying this increased sensitivity of Pglyrp2(-/-) mice to 12-O-tetradecanoylphorbol 13-acetate-induced psoriasis-like inflammation is reduced recruitment of regulatory T cells to the skin and enhanced production and activation of Th17 cells in the skin in Pglyrp2(-/-) mice, which results in more severe inflammation and keratinocyte proliferation. Thus, in wild type mice, Pglyrp2 limits overactivation of Th17 cells by promoting accumulation of regulatory T cells at the site of inflammation, which protects the skin from the exaggerated inflammatory response.

Social determinants of health impact mortality from HCC and cholangiocarcinoma: a population-based cohort study.

BACKGROUND AND AIMS: The social determinants of health can pose barriers to accessing cancer screening and treatment and have been associated with cancer mortality. However, it is not clear whether area deprivation is independently associated with mortality in HCC and cholangiocarcinoma when controlling for individual-level social determinants of health. APPROACH AND RESULTS: The cohort included individuals over 18 years old diagnosed with HCC (N=3460) or cholangiocarcinoma (N=781) and reported to the Indiana State Cancer Registry from 2009 to 2017. Area disadvantage was measured using the social deprivation index (SDI). SDI was obtained by linking addresses to the American Community Survey. Individual social determinants of health included race, ethnicity, sex, marital status, and insurance type. The primary outcome was mortality while controlling for SDI and individual social determinants of health by means of Cox proportional hazard modeling. In HCC, living in a neighborhood in the fourth quartile of census-track SDI (most deprived) was associated with higher mortality (HR: 1.14, 95% CI, 1.003-1.30, p=0.04) than living in a first quartile SDI neighborhood. Being uninsured (HR: 1.64, 95% CI, 1.30-2.07, p<0.0001) and never being married (HR: 1.31, 95% CI, 1.15-1.48, p<0.0001) were also associated with mortality in HCC. In cholangiocarcinoma, SDI was not associated with mortality. CONCLUSIONS: Social deprivation was independently associated with mortality in HCC but not cholangiocarcinoma. Further research is needed to better understand how to intervene on both area and individual social determinants of health and develop interventions to address these disparities.

ORC1 binds to cis-transcribed RNAs for efficient activation of replication origins.

Cells must coordinate the activation of thousands of replication origins dispersed throughout their genome. Active transcription is known to favor the formation of mammalian origins, although the role that RNA plays in this process remains unclear. We show that the ORC1 subunit of the human Origin Recognition Complex interacts with RNAs transcribed from genes with origins in their transcription start sites (TSSs), displaying a positive correlation between RNA binding and origin activity. RNA depletion, or the use of ORC1 RNA-binding mutant, result in inefficient activation of proximal origins, linked to impaired ORC1 chromatin release. ORC1 RNA binding activity resides in its intrinsically disordered region, involved in intra- and inter-molecular interactions, regulation by phosphorylation, and phase-separation. We show that RNA binding favors ORC1 chromatin release, by regulating its phosphorylation and subsequent degradation. Our results unveil a non-coding function of RNA as a dynamic component of the chromatin, orchestrating the activation of replication origins.

The non-catalytic role of DNA polymerase epsilon in replication initiation in human cells.

DNA polymerase epsilon (PolE) in an enzyme essential for DNA replication. Deficiencies and mutations in PolE cause severe developmental abnormalities and cancers. Paradoxically, the catalytic domain of yeast PolE catalytic subunit is dispensable for survival, and its non-catalytic essential function is linked with replicative helicase (CMG) assembly. Less is known about the PolE role in replication initiation in human cells. Here we use an auxin-inducible degron system to study the effect of POLE1 depletion on replication initiation in U2OS cells. POLE1-depleted cells were able to assemble CMG helicase and initiate DNA synthesis that failed shortly after. Expression of POLE1 non-catalytic domain rescued this defect resulting in slow, but continuous DNA synthesis. We propose a model where in human U2OS cells POLE1/POLE2 are dispensable for CMG assembly, but essential during later steps of replication initiation. Our study provides some insights into the role of PolE in replication initiation in human cells.

Loss of mismatch repair promotes a direct selective advantage in human stem cells.

Lynch syndrome (LS) is the most common hereditary form of colon cancer, resulting from a germline mutation in a DNA mismatch repair (MMR) gene. Loss of MMR in cells establishes a mutator phenotype, which may underlie its link to cancer. Acquired downstream mutations that provide the cell a selective advantage would contribute to tumorigenesis. It is unclear, however, whether loss of MMR has other consequences that would directly result in a selective advantage. We found that knockout of the MMR gene MSH2 results in an immediate survival advantage in human stem cells grown under standard cell culture conditions. This advantage results, in part, from an MMR-dependent response to oxidative stress. We also found that loss of MMR gives rise to enhanced formation and growth of human colonic organoids. These results suggest that loss of MMR may affect cells in ways beyond just increasing mutation frequency that could influence tumorigenesis.

Single-molecule imaging of replication fork conflicts at genomic DNA G4 structures in human cells.

DNA G-quadruplexes (G4s) are stable, non-canonical DNA secondary structures formed within guanine(G)-rich sequences. While extensively studied in vitro, evidence of the occurrence of G4s in vivo has only recently emerged. The formation of G4 structures may pose an obstacle for diverse DNA transactions including replication, which is linked to mutagenesis and genomic instability. A fundamental question in the field has been whether and how the formation of G4s is coupled to the progression of replication forks. This process has remained undefined largely due to the lack of experimental approaches capable of monitoring the presence of G4s and their association with the replication machinery in cells. Here, we describe a detailed multicolor single-molecule localization microscopy (SMLM) protocol for detecting nanoscale spatial-association of DNA G4s with the cellular replisome complex. This method offers a unique platform for visualizing the mechanisms of G4 formation at the molecular level, as well as addressing key biological questions as to the functional roles of these structures in the maintenance of genome integrity.