Coordination of Nucleases and Helicases during DNA Replication and Double-strand Break Repair

Nucleases and helicases are involved in numerous steps in DNA replication and repair. Nucleases act on intermediates in DNA replication created by DNA polymerases (Chapter 4) and helicases (Chapter 3). They can create substrates for repair as in Okazaki fragment processing (OFP) and homologous recombination. They can also create substrates for activation of a checkpoint response, or participate in downregulation of checkpoints. In the special case of telomere replication, they are also involved in essential processing steps (Chapter 8). Nucleases known to act during DNA replication include Dna2, Rad27, Mre11, Sae2, Exo1, RNaseH, Yen1 andMus81/Mms4. Of these, Dna2, Exo1 and Mre11 are of particular interest because they have been identified as crucial activities that initiate repair of double-strand breaks (DSBs) by homologous recombination and thus form an intrinsic link between DNA replication and repair of DSBs derived from replication fork failure. The action of the nucleases is coordinated with those of a number of helicases and is discussed here in the context of a network of their interactions that combine to maintain genome integrity during DNA replication

Coordination of Nucleases and Helicases during DNA Replication and Double-strand Break Repair

Nucleases and helicases are involved in numerous steps in DNA replication and repair. Nucleases act on intermediates in DNA replication created by DNA polymerases (Chapter 4) and helicases (Chapter 3). They can create substrates for repair as in Okazaki fragment processing (OFP) and homologous recombination. They can also create substrates for activation of a checkpoint response, or participate in downregulation of checkpoints. In the special case of telomere replication, they are also involved in essential processing steps (Chapter 8). Nucleases known to act during DNA replication include Dna2, Rad27, Mre11, Sae2, Exo1, RNaseH, Yen1 andMus81/Mms4. Of these, Dna2, Exo1 and Mre11 are of particular interest because they have been identified as crucial activities that initiate repair of double-strand breaks (DSBs) by homologous recombination and thus form an intrinsic link between DNA replication and repair of DSBs derived from replication fork failure. The action of the nucleases is coordinated with those of a number of helicases and is discussed here in the context of a network of their interactions that combine to maintain genome integrity during DNA replication

Socioeconomic inequalities in outcome of pregnancy and neonatal mortality associated with congenital anomalies: population based study

Objectives To investigate socioeconomic inequalities in outcome of pregnancy and neonatal mortality associated with congenital anomalies

Interplay of Mre11 Nuclease with Dna2 plus Sgs1 in Rad51-Dependent Recombinational Repair

The Mre11/Rad50/Xrs2 complex initiates IR repair by binding to the end of a double-strand break, resulting in 5′ to 3′ exonuclease degradation creating a single-stranded 3′ overhang competent for strand invasion into the unbroken chromosome. The nuclease(s) involved are not well understood. Mre11 encodes a nuclease, but it has 3′ to 5′, rather than 5′ to 3′ activity. Furthermore, mutations that inactivate only the nuclease activity of Mre11 but not its other repair functions, mre11-D56N and mre11-H125N, are resistant to IR. This suggests that another nuclease can catalyze 5′ to 3′ degradation. One candidate nuclease that has not been tested to date because it is encoded by an essential gene is the Dna2 helicase/nuclease. We recently reported the ability to suppress the lethality of a dna2Δ with a pif1Δ. The dna2Δ pif1Δ mutant is IR-resistant. We have determined that dna2Δ pif1Δ mre11-D56N and dna2Δ pif1Δ mre11-H125N strains are equally as sensitive to IR as mre11Δ strains, suggesting that in the absence of Dna2, Mre11 nuclease carries out repair. The dna2Δ pif1Δ mre11-D56N triple mutant is complemented by plasmids expressing Mre11, Dna2 or dna2K1080E, a mutant with defective helicase and functional nuclease, demonstrating that the nuclease of Dna2 compensates for the absence of Mre11 nuclease in IR repair, presumably in 5′ to 3′ degradation at DSB ends. We further show that sgs1Δ mre11-H125N, but not sgs1Δ, is very sensitive to IR, implicating the Sgs1 helicase in the Dna2-mediated pathway

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint

Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27^(scFEN1), encoding a 5' to 3' exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5' to 3' helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27^(ScFEN1) processes most of the Okazaki fragments, while Dna2 processes only a subset

DNA end resection by Dna2–Sgs1–RPA and its stimulation by Top3–Rmi1 and Mre11–Rad50–Xrs2

The repair of DNA double-strand breaks (DSBs) by homologous recombination requires processing of broken ends. For repair to start, the DSB must first be resected to generate a 3′-single-stranded DNA (ssDNA) overhang, which becomes a substrate for the DNA strand exchange protein, Rad51 (ref. 1). Genetic studies have implicated a multitude of proteins in the process, including helicases, nucleases and topoisomerases. Here we biochemically reconstitute elements of the resection process and reveal that it requires the nuclease Dna2, the RecQ-family helicase Sgs1 and the ssDNA-binding protein replication protein-A (RPA). We establish that Dna2, Sgs1 and RPA constitute a minimal protein complex capable of DNA resection in vitro. Sgs1 helicase unwinds the DNA to produce an intermediate that is digested by Dna2, and RPA stimulates DNA unwinding by Sgs1 in a species-specific manner. Interestingly, RPA is also required both to direct Dna2 nucleolytic activity to the 5′-terminated strand of the DNA break and to inhibit 3′ to 5′ degradation by Dna2, actions that generate and protect the 3′-ssDNA overhang, respectively. In addition to this core machinery, we establish that both the topoisomerase 3 (Top3) and Rmi1 complex and the Mre11–Rad50–Xrs2 complex (MRX) have important roles as stimulatory components. Stimulation of end resection by the Top3–Rmi1 heterodimer and the MRX proteins is by complex formation with Sgs1 (refs 5, 6), which unexpectedly stimulates DNA unwinding. We suggest that Top3–Rmi1 and MRX are important for recruitment of the Sgs1–Dna2 complex to DSBs. Our experiments provide a mechanistic framework for understanding the initial steps of recombinational DNA repair in eukaryotes

Socioeconomic inequalities in pregnancy outcome associated with Down syndrome: a population-based study.

OBJECTIVE: To investigate socioeconomic inequalities in outcome of pregnancy associated with Down syndrome (DS) compared with other congenital anomalies screened for during pregnancy. DESIGN AND SETTING: Retrospective population-based registry study (East Midlands & South Yorkshire in England). PARTICIPANTS: All registered cases of DS and nine selected congenital anomalies with poor prognostic outcome (the UK Fetal Anomaly Screening Programme (FASP)9) with an end of pregnancy date between 1 January 1998 and 31 December 2007. MAIN OUTCOME MEASURES: Poisson regression models were used to explore outcome measures, including socioeconomic variation in rates of anomaly; antenatal detection; pregnancy outcome; live birth incidence and neonatal mortality. Deprivation was measured using the Index of Multiple Deprivation 2004 at super output area level. RESULTS: There were 1151 cases of DS and 1572 cases of the nine severe anomalies combined. The overall rate of antenatal detection was 57% for DS, which decreased with increasing deprivation (rate ratio comparing the most deprived tenth with the least deprived: 0.76 (0.60 to 0.97)). Antenatal detection rates were considerably higher for FASP9 anomalies (86%), with no evidence of a trend with deprivation (0.99 95% CI (0.84 to 1.17)). The termination of pregnancy rate following antenatal diagnosis was higher for DS (86%) than the FASP9 anomalies (70%). Both groups showed wide socioeconomic variation in the termination of pregnancy rate (rate ratio: DS: 0.76 (0.58 to 0.99); FASP9 anomalies: 0.80 (0.65 to 0.97)). Consequently, socioeconomic inequalities in live birth and neonatal mortality rates associated with these anomalies arise that were not observed in utero. CONCLUSIONS: Socioeconomic inequalities exist in the antenatal detection of DS, and subsequent termination rates are much higher for DS than other anomalies. Termination rates for all anomalies are lower in more deprived areas leading to wide socioeconomic inequalities in live born infants with a congenital anomaly, particularly DS, and subsequent neonatal mortality

The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo

In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Δ cells. Hence, we conclude that CEB1 instability in pif1Δ cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK.

BACKGROUND: A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. METHODS: This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. FINDINGS: Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0-75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4-97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8-80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3-4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. INTERPRETATION: ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, Bill & Melinda Gates Foundation, Lemann Foundation, Rede D'Or, Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca