To skip or not to skip: choosing repriming to tolerate DNA damage.

Accurate DNA replication is constantly threatened by DNA lesions arising from endogenous and exogenous sources. Specialized DNA replication stress response pathways ensure replication fork progression in the presence of DNA lesions with minimal delay in fork elongation. These pathways broadly include translesion DNA synthesis, template switching, and replication fork repriming. Here, we discuss recent advances toward our understanding of the mechanisms that regulate the fine-tuned balance between these different replication stress response pathways. We also discuss the molecular pathways required to fill single-stranded DNA gaps that accumulate throughout the genome after repriming and the biological consequences of using repriming instead of other DNA damage tolerance pathways on genome integrity and cell fitness.

Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells.

PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLζ translesion synthesis polymerases promotes gap filling in G2. The E2-conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLζ are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.

The SMC5/6 complex prevents genotoxicity upon APOBEC3A-mediated replication stress.

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.

A RAD18-UBC13-PALB2-RNF168 axis mediates replication fork recovery in BRCA1-deficient cancer cells.

BRCA1/2 proteins function in genome stability by promoting repair of double-stranded DNA breaks through homologous recombination and by protecting stalled replication forks from nucleolytic degradation. In BRCA1/2-deficient cancer cells, extensively degraded replication forks can be rescued through distinct fork recovery mechanisms that also promote cell survival. Here, we identified a novel pathway mediated by the E3 ubiquitin ligase RAD18, the E2-conjugating enzyme UBC13, the recombination factor PALB2, the E3 ubiquitin ligase RNF168 and PCNA ubiquitination that promotes fork recovery in BRCA1- but not BRCA2-deficient cells. We show that this pathway does not promote fork recovery by preventing replication fork reversal and degradation in BRCA1-deficient cells. We propose a mechanism whereby the RAD18-UBC13-PALB2-RNF168 axis facilitates resumption of DNA synthesis by promoting re-annealing of the complementary single-stranded template strands of the extensively degraded forks, thereby allowing re-establishment of a functional replication fork. We also provide preliminary evidence for the potential clinical relevance of this novel fork recovery pathway in BRCA1-mutated cancers, as RAD18 is over-expressed in BRCA1-deficient cancers, and RAD18 loss compromises cell viability in BRCA1-deficient cancer cells.

PRIMPOL ready, set, reprime!

DNA replication forks are constantly challenged by DNA lesions induced by endogenous and exogenous sources. DNA damage tolerance mechanisms ensure that DNA replication continues with minimal effects on replication fork elongation either by using specialized DNA polymerases, which have the ability to replicate through the damaged template, or by skipping the damaged DNA, leaving it to be repaired after replication. These mechanisms are evolutionarily conserved in bacteria, yeast, and higher eukaryotes, and are paramount to ensure timely and faithful duplication of the genome. The Primase and DNA-directed Polymerase (PRIMPOL) is a recently discovered enzyme that possesses both primase and polymerase activities. PRIMPOL is emerging as a key player in DNA damage tolerance, particularly in vertebrate and human cells. Here, we review our current understanding of the function of PRIMPOL in DNA damage tolerance by focusing on the structural aspects that define its dual enzymatic activity, as well as on the mechanisms that control its chromatin recruitment and expression levels. We also focus on the latest findings on the mitochondrial and nuclear functions of PRIMPOL and on the impact of loss of these functions on genome stability and cell survival. Defining the function of PRIMPOL in DNA damage tolerance is becoming increasingly important in the context of human disease. In particular, we discuss recent evidence pointing at the PRIMPOL pathway as a novel molecular target to improve cancer cell response to DNA-damaging chemotherapy and as a predictive parameter to stratify patients in personalized cancer therapy.

RNase H activities counteract a toxic effect of Polymerase η in cells replicating with depleted dNTP pools.

RNA:DNA hybrids are transient physiological intermediates that arise during several cellular processes such as DNA replication. In pathological situations, they may stably accumulate and pose a threat to genome integrity. Cellular RNase H activities process these structures to restore the correct DNA:DNA sequence. Yeast cells lacking RNase H are negatively affected by depletion of deoxyribonucleotide pools necessary for DNA replication. Here we show that the translesion synthesis DNA polymerase η (Pol η) plays a role in DNA replication under low deoxyribonucleotides condition triggered by hydroxyurea. In particular, the catalytic reaction performed by Pol η is detrimental for RNase H deficient cells, causing DNA damage checkpoint activation and G2/M arrest. Moreover, a Pol η mutant allele with enhanced ribonucleotide incorporation further exacerbates the sensitivity to hydroxyurea of cells lacking RNase H activities. Our data are compatible with a model in which Pol η activity facilitates the formation or stabilization of RNA:DNA hybrids at stalled replication forks. However, in a scenario where RNase H activity fails to restore DNA, these hybrids become highly toxic for cells.

The Incorporation of Ribonucleotides Induces Structural and Conformational Changes in DNA.

Ribonucleotide incorporation is the most common error occurring during DNA replication. Cells have hence developed mechanisms to remove ribonucleotides from the genome and restore its integrity. Indeed, the persistence of ribonucleotides into DNA leads to severe consequences, such as genome instability and replication stress. Thus, it becomes important to understand the effects of ribonucleotides incorporation, starting from their impact on DNA structure and conformation. Here we present a systematic study of the effects of ribonucleotide incorporation into DNA molecules. We have developed, to our knowledge, a new method to efficiently synthesize long DNA molecules (hundreds of basepairs) containing ribonucleotides, which is based on a modified protocol for the polymerase chain reaction. By means of atomic force microscopy, we could therefore investigate the changes, upon ribonucleotide incorporation, of the structural and conformational properties of numerous DNA populations at the single-molecule level. Specifically, we characterized the scaling of the contour length with the number of basepairs and the scaling of the end-to-end distance with the curvilinear distance, the bending angle distribution, and the persistence length. Our results revealed that ribonucleotides affect DNA structure and conformation on scales that go well beyond the typical dimension of the single ribonucleotide. In particular, the presence of ribonucleotides induces a systematic shortening of the molecules, together with a decrease of the persistence length. Such structural changes are also likely to occur in vivo, where they could directly affect the downstream DNA transactions, as well as interfere with protein binding and recognition.

DNA combing versus DNA spreading and the separation of sister chromatids.

DNA combing and DNA spreading are two central approaches for studying DNA replication fork dynamics genome-wide at single-molecule resolution by distributing labeled genomic DNA on coverslips or slides for immunodetection. Perturbations in DNA replication fork dynamics can differentially affect either leading or lagging strand synthesis, for example, in instances where replication is blocked by a lesion or obstacle on only one of the two strands. Thus, we sought to investigate whether the DNA combing and/or spreading approaches are suitable for resolving adjacent sister chromatids during DNA replication, thereby enabling the detection of DNA replication dynamics within individual nascent strands. To this end, we developed a thymidine labeling scheme that discriminates between these two possibilities. Our data suggests that DNA combing resolves sister chromatids, allowing the detection of strand-specific alterations, whereas DNA spreading typically does not. These findings have important implications when interpreting DNA replication dynamics from data obtained by these two commonly used techniques.

The SMC5/6 complex prevents genotoxicity upon APOBEC3A-mediated replication stress.

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.

DNA Combing versus DNA Spreading and the Separation of Sister Chromatids.

DNA combing and DNA spreading are two central approaches for studying DNA replication fork dynamics genome-wide at single-molecule resolution by distributing labeled genomic DNA on coverslips or slides for immunodetection. Perturbations in DNA replication fork dynamics can differentially affect either leading or lagging strand synthesis, for example in instances where replication is blocked by a lesion or obstacle on only one of the two strands. Thus, we sought to investigate whether the DNA combing and/or spreading approaches are suitable for resolving adjacent sister chromatids during DNA replication, thereby enabling the detection of DNA replication dynamics within individual nascent strands. To this end, we developed a thymidine labeling scheme that discriminates between these two possibilities. Our data suggests that DNA combing resolves single chromatids, allowing the detection of strand-specific alterations, whereas DNA spreading does not. These findings have important implications when interpreting DNA replication dynamics from data obtained by these two commonly used techniques.

Rapid profiling of DNA replication dynamics using mass spectrometry-based analysis of nascent DNA.

The primary method for probing DNA replication dynamics is DNA fiber analysis, which utilizes thymidine analog incorporation into nascent DNA, followed by immunofluorescent microscopy of DNA fibers. Besides being time-consuming and prone to experimenter bias, it is not suitable for studying DNA replication dynamics in mitochondria or bacteria, nor is it adaptable for higher-throughput analysis. Here, we present mass spectrometry-based analysis of nascent DNA (MS-BAND) as a rapid, unbiased, quantitative alternative to DNA fiber analysis. In this method, incorporation of thymidine analogs is quantified from DNA using triple quadrupole tandem mass spectrometry. MS-BAND accurately detects DNA replication alterations in both the nucleus and mitochondria of human cells, as well as bacteria. The high-throughput capability of MS-BAND captured replication alterations in an E. coli DNA damage-inducing gene library. Therefore, MS-BAND may serve as an alternative to the DNA fiber technique, with potential for high-throughput analysis of replication dynamics in diverse model systems.

A RADAR method to measure DNA topoisomerase covalent complexes.

DNA topoisomerases resolve topological stress by introducing transient single- or double-strand breaks into the DNA duplex. This reaction requires the covalent binding of topoisomerases to DNA while the topological stress is being released. This transient intermediate is known as topoisomerase-covalent complex and represents the target of many anti-cancer drugs. Here, we describe a protocol to quantitatively detect topoisomerase-covalent complexes in vivo, called RADAR (rapid approach to DNA adduct recovery). DNA and protein-DNA covalent complexes are rapidly isolated from cells through chaotropic extraction. After normalization, samples are loaded on a slot blot, and the covalent complexes are detected using specific topoisomerase antibodies. In addition to being fast and robust, this assay produces quantitative results. Consequently, the RADAR assay can be applied to investigate the topoisomerase-covalent complex biology, including the effect of specific topoisomerase inhibitors. Finally, the same assay can be more generally applied to study covalent complexes of other enzymes with DNA.

NEDDylated Cullin 3 mediates the adaptive response to topoisomerase 1 inhibitors.

DNA topoisomerase 1 (TOP11) inhibitors are mainstays of anticancer therapy. These drugs trap TOP1 on DNA, stabilizing the TOP1-cleavage complex (TOP1-cc). The accumulation of TOP1-ccs perturbs DNA replication fork progression, leading to DNA breaks and cell death. By analyzing the genomic occupancy and activity of TOP1, we show that cells adapt to treatment with multiple doses of TOP1 inhibitor by promoting the degradation of TOP1-ccs, allowing cells to better tolerate subsequent doses of TOP1 inhibitor. The E3-RING Cullin 3 ligase in complex with the BTBD1 and BTBD2 adaptor proteins promotes TOP1-cc ubiquitination and subsequent proteasomal degradation. NEDDylation of Cullin 3 activates this pathway, and inhibition of protein NEDDylation or depletion of Cullin 3 sensitizes cancer cells to TOP1 inhibitors. Collectively, our data uncover a previously unidentified NEDD8-Cullin 3 pathway involved in the adaptive response to TOP1 inhibitors, which can be targeted to improve the efficacy of TOP1 drugs in cancer therapy.

Maternal Dietary Carbohydrate Intake and Newborn Aortic Wall Thickness.

Evidence from animal models indicates that maternal diet during pregnancy affects offspring cardiometabolic health. Improving carbohydrate quality during high-risk pregnancies reduces aortic intima-medial thickness; a marker for early atherosclerosis; in the infant offspring. We sought to determine whether maternal carbohydrate quantity and quality are associated with newborn aortic intima-medial thickness in healthy pregnancies. Maternal diet throughout pregnancy was evaluated in 139 mother-child dyads using a validated food frequency questionnaire. Carbohydrate intake was expressed as quantity (% total energy), quality (fibre, glycaemic index), and glycaemic burden (glycaemic load). Aortic intima-medial thickness was measured by high-frequency ultrasound of the neonatal abdominal aorta. Neither quantity nor quality of maternal carbohydrate intake during pregnancy was associated with meaningful differences in offspring maximum aortic intima-medial thickness with the exception of fibre intake in women with overweight or obesity which was inversely associated (-8 µm [95% CI -14, -1] per g fibre, p = 0.04). In healthy pregnancy, the quantity and quality of maternal carbohydrate intake is likely not a meaningful modifiable lifestyle factor for influencing offspring vascular health. The effect of carbohydrate quality may only be evident in high-risk pregnancies, consistent with previous findings. These findings may be confirmed in prospective dietary trials in pregnancy.

ISG15 fast-tracks DNA replication.

In this issue, Raso et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.202002175) uncover a novel replication fork speed regulatory network controlled by the ubiquitin-like modifier interferon-stimulated gene 15 (ISG15), which plays a central role in the innate immune response and regulates tumorigenesis as well as chemotherapy response.

Is a Higher Protein-Lower Glycemic Index Diet More Nutritious Than a Conventional Diet? A PREVIEW Sub-study.

High protein diets and low glycemic index (GI) diets have been associated with improved diet quality. We compared the changes in nutrient intakes of individuals at high risk of developing type-2 diabetes over 3 y who followed either a higher protein-lower GI diet (HPLG) or a conventional moderate protein-moderate GI diet (MPMG). This post hoc analysis included 161 participants with overweight and pre-diabetes from the Australian cohort of the PREVIEW study (clinical trial registered in https://www.clinicaltrials.gov/ct2/show/NCT01777893?term=NCT01777893&draw=2&rank=1) who were randomly assigned to a HPLG diet (25% energy from protein, dietary GI ≤ 50, n = 85) or a MPMG diet (15% energy from protein, dietary GI ≥ 56, n = 76). Food records were collected at 0-mo (baseline) and at 6-, 12-, 24-, and 36-mo (dietary intervention period). Linear mixed models were used to compare the differences in total energy, macro- and micronutrients, dietary GI, glycemic load (GL) and body weight between the two diet groups at the 4 dietary intervention time points. At 3 y, 74% participants from the HPLG diet and 74% participants from the MPMG diet completed the trial. The HPLG group showed significantly higher protein intake and lower dietary GI and GL than the MPMG group (group fixed effect P < 0.001 for all three parameters). By 6-, 12-, 24-, and 36-mo there was a 3.0, 2.7, 2.2, and 1.4% point difference in protein intake and 6.2, 4.1, 4.8, and 3.9 GI unit difference between the groups. The intake of energy and saturated fat decreased (mostly in the first 6-mo), while the intake of dietary fiber increased (from mo-0 to mo-12 only) in both diets, with no significant differences between the diets. The dietary intakes of zinc (group fixed effect P = 0.05), selenium (P = 0.01), niacin (P = 0.01), vitamin B12 (P = 0.01) and dietary cholesterol (group by time fixed effect P = 0.001) were higher in the HPLG group than in the MPMG group. Despite both diets being designed to be nutritionally complete, a HPLG diet was found to be more nutritious in relation to some micronutrients, but not cholesterol, than a MPMG diet.

Validation of a cuff-based device for measuring carotid-femoral pulse wave velocity in children and adolescents.

Carotid-femoral pulse wave velocity is associated with arterial stiffness in major elastic arteries, and predicts future cardiovascular events. However, little is known about carotid-femoral pulse wave velocity as a marker of vascular health in children. Semi-automated cuff-based devices for assessing pulse wave velocity are increasingly popular, although these utilize an algorithm developed and validated in adults. Physiological differences between adults and children may, however, reduce the accuracy of cuff-based methods. We sought to determine the accuracy of a cuff-based pulse wave velocity device in healthy children, and determine whether a novel age-appropriate algorithm increases accuracy. We recruited 29 healthy children between the ages of 2 and 20 years. Pulse wave velocity was measured both by using a tonometer on the carotid artery and an inflated cuff on the thigh, and using a tonometer on both the carotid artery and femoral artery as a reference standard. Accuracy of the cuff-based device with its standard algorithm developed in adults, and a novel age-appropriate algorithm corrected for physiological differences in leg pulse wave velocity was assessed with Regression analysis and Bland-Altman plots. Cuff-based device estimates of pulse wave velocity had excellent agreement to the reference standard (Δ = -0.26 ms-1 [SD 0.44]). The novel age-appropriate algorithm improved the accuracy of the cuff-based method (Δ = 0.02 ms-1 [SD 0.44]). The cuff-based semi-automatic approach estimates carotid-femoral pulse wave velocity with excellent agreement to the reference standard. However, adjusting the algorithm for known differences in leg pulse wave velocity further improves the accuracy of cuff-based measurement in children and adolescents.

'Boden Food Plate': Novel Interactive Web-based Method for the Assessment of Dietary Intake.

Different methods can be used in research to assess dietary intake, many of which are still paper-based. Written estimated food diaries are often utilized in clinical trials, despite being a burden for both study participants and researchers. This method requires participant literacy, it is time consuming, labor intensive, and can easily lead to under-reporting. With advancements in technology, there is a growing interest in electronic diaries that automate the dietary assessment process. These are focused on improving accuracy, reducing both time and cost and providing users with a visual and more enjoyable experience. The methodology presented here aimed to validate the 'Boden Food Plate', a novel web-based platform for self-recording of food and drink items, compared to a traditional estimated food diary. The application was also rated on a satisfaction scale by study participants using a paper-based questionnaire. Sixty-seven participants completed the dietary measures on both the three-day electronic and paper food diaries. For the analysis, only dietary data completed at both study time points (baseline and week six) was utilized. Despite small mean differences between dietary data collection methods, Bland Altman analysis showed fairly wide 95% limits of agreement between the electronic platform and the written estimated food diary and there were few cases which did not fall within the 95% confidence intervals. Overall, participants found the electronic food diary to be more fun than the paper method and as easy to use as hard copy diaries. The new platform has potential as a self-recording tool for the collection of dietary data, particularly when utilized in clinical trial settings. However, further validation studies are needed to improve the validity of this novel electronic dietary data collection tool.

Characterization of Structural and Configurational Properties of DNA by Atomic Force Microscopy.

We describe a method to extract quantitative information on DNA structural and configurational properties from high-resolution topographic maps recorded by atomic force microscopy (AFM). DNA molecules are deposited on mica surfaces from an aqueous solution, carefully dehydrated, and imaged in air in Tapping Mode. Upon extraction of the spatial coordinates of the DNA backbones from AFM images, several parameters characterizing DNA structure and configuration can be calculated. Here, we explain how to obtain the distribution of contour lengths, end-to-end distances, and gyration radii. This modular protocol can be also used to characterize other statistical parameters from AFM topographies.

Measuring the Levels of Ribonucleotides Embedded in Genomic DNA.

Ribonucleotides (rNTPs) are incorporated into genomic DNA at a relatively high frequency during replication. They have beneficial effects but, if not removed from the chromosomes, increase genomic instability. Here, we describe a fast method to easily estimate the amounts of embedded ribonucleotides into the genome. The protocol described is performed in Saccharomyces cerevisiae and allows us to quantify altered levels of rNMPs due to different mutations in the replicative polymerase ε. However, this protocol can be easily applied to cells derived from any organism.