scholarly journals Genetic Study of Four Candidate Holliday Junction Processing Proteins in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius

2022 ◽  
Vol 23 (2) ◽  
pp. 707
Author(s):  
Ryo Matsuda ◽  
Shoji Suzuki ◽  
Norio Kurosawa
Keyword(s):  
Functional Relation ◽  
Cellular Growth ◽  
Sulfolobus Acidocaldarius ◽  
Hyperthermophilic Archaea ◽  
Holliday Junction ◽  
Cold Sensitivity ◽  
Replication Forks ◽  
Double Knockout ◽  
Stalled Replication Forks

Homologous recombination (HR) is thought to be important for the repair of stalled replication forks in hyperthermophilic archaea. Previous biochemical studies identified two branch migration helicases (Hjm and PINA) and two Holliday junction (HJ) resolvases (Hjc and Hje) as HJ-processing proteins; however, due to the lack of genetic evidence, it is still unclear whether these proteins are actually involved in HR in vivo and how their functional relation is associated with the process. To address the above questions, we constructed hjc-, hje-, hjm-, and pina single-knockout strains and double-knockout strains of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. Notably, we succeeded in isolating the hjm- and/or pina-deleted strains, suggesting that the functions of Hjm and PINA are not essential for cellular growth in this archaeon, as they were previously thought to be essential. Growth retardation in Δpina was observed at low temperatures (cold sensitivity). When deletion of the HJ resolvase genes was combined, Δpina Δhjc and Δpina Δhje exhibited severe cold sensitivity. Δhjm exhibited severe sensitivity to interstrand crosslinkers, suggesting that Hjm is involved in repairing stalled replication forks, as previously demonstrated in euryarchaea. Our findings suggest that the function of PINA and HJ resolvases is functionally related at lower temperatures to support robust cellular growth, and Hjm is important for the repair of stalled replication forks in vivo.

scholarly journals Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

2015 ◽  
Vol 197 (17) ◽  
pp. 2792-2809 ◽  
Author(s):  
Sarita Mallik ◽  
Ellen M. Popodi ◽  
Andrew J. Hanson ◽  
Patricia L. Foster
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Replication Forks ◽  
Content Type ◽  
Pol Iv ◽  
Dna Polymerase Iv ◽  
Stalled Replication Forks

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

scholarly journals Regulation of Rtt107 Recruitment to Stalled DNA Replication Forks by the Cullin Rtt101 and the Rtt109 Acetyltransferase

2008 ◽  
Vol 19 (1) ◽  
pp. 171-180 ◽  
Author(s):  
Tania M. Roberts ◽  
Iram Waris Zaidi ◽  
Jessica A. Vaisica ◽  
Matthias Peter ◽  
Grant W. Brown
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Replication Forks ◽  
Chromatin Binding ◽  
Direct Role ◽  
Repair Enzymes ◽  
Damage Response ◽  
Stalled Replication Forks

RTT107 (ESC4, YHR154W) encodes a BRCA1 C-terminal domain protein that is important for recovery from DNA damage during S phase. Rtt107 is a substrate of the checkpoint kinase Mec1, and it forms complexes with DNA repair enzymes, including the nuclease subunit Slx4, but the role of Rtt107 in the DNA damage response remains unclear. We find that Rtt107 interacts with chromatin when cells are treated with compounds that cause replication forks to arrest. This damage-dependent chromatin binding requires the acetyltransferase Rtt109, but it does not require acetylation of the known Rtt109 target, histone H3-K56. Chromatin binding of Rtt107 also requires the cullin Rtt101, which seems to play a direct role in Rtt107 recruitment, because the two proteins are found in complex with each other. Finally, we provide evidence that Rtt107 is bound at or near stalled replication forks in vivo. Together, these results indicate that Rtt109, Rtt101, and Rtt107, which genetic evidence suggests are functionally related, form a DNA damage response pathway that recruits Rtt107 complexes to damaged or stalled replication forks.

scholarly journals Colocalization of Mec1 and Mrc1 is sufficient for Rad53 phosphorylation in vivo

2012 ◽  
Vol 23 (6) ◽  
pp. 1058-1067 ◽  
Author(s):  
Theresa J. Berens ◽  
David P. Toczyski
Keyword(s):  
Dna Replication ◽  
Double Strand Breaks ◽  
Sensor Kinase ◽  
Replication Forks ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Strand Breaks ◽  
Crucial Step ◽  
Stalled Replication Forks

When DNA is damaged or DNA replication goes awry, cells activate checkpoints to allow time for damage to be repaired and replication to complete. In Saccharomyces cerevisiae, the DNA damage checkpoint, which responds to lesions such as double-strand breaks, is activated when the lesion promotes the association of the sensor kinase Mec1 and its targeting subunit Ddc2 with its activators Ddc1 (a member of the 9-1-1 complex) and Dpb11. It has been more difficult to determine what role these Mec1 activators play in the replication checkpoint, which recognizes stalled replication forks, since Dpb11 has a separate role in DNA replication itself. Therefore we constructed an in vivo replication-checkpoint mimic that recapitulates Mec1-dependent phosphorylation of the effector kinase Rad53, a crucial step in checkpoint activation. In the endogenous replication checkpoint, Mec1 phosphorylation of Rad53 requires Mrc1, a replisome component. The replication-checkpoint mimic requires colocalization of Mrc1-LacI and Ddc2-LacI and is independent of both Ddc1 and Dpb11. We show that these activators are also dispensable for Mec1 activity and cell survival in the endogenous replication checkpoint but that Ddc1 is absolutely required in the absence of Mrc1. We propose that colocalization of Mrc1 and Mec1 is the minimal signal required to activate the replication checkpoint.

scholarly journals PrimPol is required for replication reinitiation after mtDNA damage

2017 ◽  
Vol 114 (43) ◽  
pp. 11398-11403 ◽  
Author(s):  
Rubén Torregrosa-Muñumer ◽  
Josefin M. E. Forslund ◽  
Steffi Goffart ◽  
Annika Pfeiffer ◽  
Gorazd Stojkovič ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Translesion Synthesis ◽  
Replication Forks ◽  
Mtdna Damage ◽  
Replicative Stress ◽  
Mtdna Replication ◽  
Stalled Replication Forks ◽  
Lagging Strand

Eukaryotic PrimPol is a recently discovered DNA-dependent DNA primase and translesion synthesis DNA polymerase found in the nucleus and mitochondria. Although PrimPol has been shown to be required for repriming of stalled replication forks in the nucleus, its role in mitochondria has remained unresolved. Here we demonstrate in vivo and in vitro that PrimPol can reinitiate stalled mtDNA replication and can prime mtDNA replication from nonconventional origins. Our results not only help in the understanding of how mitochondria cope with replicative stress but can also explain some controversial features of the lagging-strand replication.

scholarly journals The Nse5-Nse6 Dimer Mediates DNA Repair Roles of the Smc5-Smc6 Complex

2006 ◽  
Vol 26 (5) ◽  
pp. 1617-1630 ◽  
Author(s):  
Stephanie Pebernard ◽  
James Wohlschlegel ◽  
W. Hayes McDonald ◽  
John R. Yates ◽  
Michael N. Boddy
Keyword(s):  
Dna Repair ◽  
Holliday Junctions ◽  
Holliday Junction ◽  
Mitotic Catastrophe ◽  
Replication Forks ◽  
Uv Sensitivity ◽  
Recombination Repair ◽  
High Level ◽  
Stalled Replication Forks ◽  
Uv Lesions

ABSTRACT Stabilization and processing of stalled replication forks is critical for cell survival and genomic integrity. We characterize a novel DNA repair heterodimer of Nse5 and Nse6, which are nonessential nuclear proteins critical for chromosome segregation in fission yeast. The Nse5/6 dimer facilitates DNA repair as part of the Smc5-Smc6 holocomplex (Smc5/6), the basic architecture of which we define. Nse5-Nes6 (Nse5/6) mutants display a high level of spontaneous DNA damage and mitotic catastrophe in the absence of the master checkpoint regulator Rad3 (hATR). Nse5/6 mutants are required for the response to genotoxic agents that block the progression of replication forks, acting in a pathway that allows the tolerance of irreparable UV lesions. Interestingly, the UV sensitivity of Nse5/6 mutants is suppressed by concomitant deletion of the homologous recombination repair factor, Rhp51 (Rad51). Further, the viability of Nse5/6 mutants depends on Mus81 and Rqh1, factors that resolve or prevent the formation of Holliday junctions. Consistently, the UV sensitivity of cells lacking Nse5/6 can be partially suppressed by overexpressing the bacterial resolvase RusA. We propose a role for Nse5/6 mutants in suppressing recombination that results in Holliday junction formation or in Holliday junction resolution.

scholarly journals Dimerization of the ATRIP Protein through the Coiled-Coil Motif and Its Implication to the Maintenance of Stalled Replication Forks

2005 ◽  
Vol 16 (12) ◽  
pp. 5551-5562 ◽  
Author(s):  
Eisuke Itakura ◽  
Isao Sawada ◽  
Akira Matsuura
Keyword(s):  
Mammalian Cells ◽  
Coiled Coil ◽  
Genotoxic Stress ◽  
Replication Forks ◽  
Cycle Arrest ◽  
Coiled Coil Domain ◽  
Functional Complex ◽  
Stalled Replication Forks

ATR (ATM and Rad3-related), a PI kinase-related kinase (PIKK), has been implicated in the DNA structure checkpoint in mammalian cells. ATR associates with its partner protein ATRIP to form a functional complex in the nucleus. In this study, we investigated the role of the ATRIP coiled-coil domain in ATR-mediated processes. The coiled-coil domain of human ATRIP contributes to self-dimerization in vivo, which is important for the stable translocation of the ATR-ATRIP complex to nuclear foci that are formed after exposure to genotoxic stress. The expression of dimerization-defective ATRIP diminishes the maintenance of replication forks during treatment with replication inhibitors. By contrast, it does not compromise the G2/M checkpoint after IR-induced DNA damage. These results show that there are two critical functions of ATR-ATRIP after the exposure to genotoxic stress: maintenance of the integrity of replication machinery and execution of cell cycle arrest, which are separable and are achieved via distinct mechanisms. The former function may involve the concentrated localization of ATR to damaged sites for which the ATRIP coiled-coil motif is critical.

The Bacillus subtilis PriA winged helix domain is critical for surviving DNA damage

2022 ◽  
Author(s):  
Lindsay A. Matthews ◽  
Lyle A. Simmons
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Replication Fork ◽  
Pathogenic Bacteria ◽  
Replication Forks ◽  
Human Pathogens ◽  
Winged Helix ◽  
E Coli ◽  
Stalled Replication Forks

DNA replication forks regularly encounter lesions or other impediments that result in a blockage to fork progression. PriA is one of the key proteins used by virtually all eubacteria to survive conditions that result in a blockage to replication fork movement. PriA directly binds stalled replication forks and initiates fork restart allowing for chromosomes to be fully duplicated under stressful conditions. We used a CRISPR-Cas gene editing approach to map PriA residues critical for surviving DNA damage induced by several antibiotics in B. subtilis . We find that the winged helix (WH) domain in B. subtilis PriA is critical for surviving DNA damage and participates in DNA binding. The critical in vivo function of the WH domain mapped to distinct surfaces that were also conserved among several Gram-positive human pathogens. In addition, we identified an amino acid linker neighboring the WH domain that is greatly extended in B. subtilis due to an insertion. Shortening this linker induced a hypersensitive phenotype to DNA damage, suggesting that its extended length is critical for efficient replication fork restart in vivo . Because the WH domain is dispensable in E. coli PriA, our findings demonstrate an important difference in the contribution of the WH domain during fork restart in B. subtilis . Further, with our results we suggest that this highly variable region in PriA could provide different functions across diverse bacterial organisms. IMPORTANCE PriA is an important protein found in virtually all bacteria that recognizes stalled replication forks orchestrating fork restart. PriA homologs contain a winged helix (WH) domain which is dispensable in E. coli and functions in a fork restart pathway that is not conserved outside of E. coli and closely related proteobacteria. We analyzed the importance of the WH domain and an associated linker in B. subtilis and found that both are critical for surviving DNA damage. This function mapped to a small motif at the C-terminal end of the WH domain, which is also conserved in pathogenic bacteria. The motif was not required for DNA binding and therefore may perform a novel function in the replication fork restart pathway.

scholarly journals PARP Inhibitor Veliparib and Busulfan in a Xenograft Model of Myeloproliferative Neoplasm

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3319-3319
Author(s):  
Pritesh Patel ◽  
Vitalyi Senyuk ◽  
Natalie S. Rodriguez ◽  
Dolores Mahmud ◽  
Annie Lee Oh ◽  
...  
Keyword(s):  
High Risk ◽  
Combination Treatment ◽  
Xenograft Model ◽  
Replication Forks ◽  
Cd34 Cells ◽  
Synergistic Cytotoxicity ◽  
Hel Cells ◽  
Stalled Replication Forks ◽  
Parp 1

Abstract Despite new targeted therapies, patients with high risk myeloproliferative neoplasms (MPNs) have an increased chance of transforming into acute myeloid leukemia (AML) and can only be cured with allogeneic hematopoietic stem cell transplant (HSCT). JAK2V617F mutation is common in MPNs and is associated with genomic instability, with baseline DNA double strand breaks and homologous recombination (HR) activity as a result of stalled replication forks. Poly(ADP-ribose) polymerase-1 (PARP-1) detects disrupted replication forks and recruits HR repair enzymes to restart DNA replication. Because busulfan, which is used in preparative regimens for HSCT, also leads to stalled replication forks through DNA strand crosslinking, we hypothesized that the PARP-1 inhibitor veliparib and busulfan may lead to synergistic cytotoxicity in MPN cells. We first treated two JAK2V617F positive MPN cell lines with increasing doses (from 0.1 to 100μM) of veliparib in liquid cultures and measured cell proliferation. SET2 and HEL cells were relatively sensitive to veliparib (IC50 of 11.3μM and 74.2μM respectively). We next treated the cell lines with increasing doses of busulfan in combination with a fixed subtherapeutic dose of veliparib (4μM) for 48 hours. With combination treatment, the busulfan IC50 decreased from 27μM to 4μM in SET2 cells and from 45.1μM to 28.1μM in HEL cells. The combination was synergistic with a mean combination index (CI) of 0.55 for SET2 and 0.40 for HEL cells. Combination treatment resulted in a higher fraction of cells in G2M arrest than with veliparib alone (p=0.03) or busulfan alone (p=0.04). Similarly, when the combination treatment was tested on CD34+ cells obtained from 5 patients with JAK2V617F mutated and 2 CALR mutated PMF in a standard clonogenic (CFU) assay, it caused a greater inhibition of colony formation than busulfan or veliparib alone (p=0.001). Veliparib alone did not show any effect on colony formation from healthy control CD34+ cells. Finally we utilized a xenograft model with NOD/SCID/IL-2Rγnull (NSG) mice to test the in-vivo effect of combined low doses of busulfan and veliparib in JAK2V617F MPNs. In order to establish disease, mice were injected with 5x106 SET2 cells via tail vein 12 hours after sub-lethal irradiation. Then, 14 days after SET-2 injection 4 groups of mice (n=5 each) were treated for up to 3 weeks with intra-peritoneal vehicle (control); or a subtherapeutic dose of veliparib (3mg/kg daily) for 5 days a week; or busulfan (25mg/kg weekly); or a combination of both drugs. Death from leukemia was documented by marrow and spleen cell immunophenotype using non-cross reactive anti-human CD33 and CD34 antibodies. Veliparib alone did not improve survival vs control. On the contrary, survival was increased by the combination treatment vs busulfan alone (p=0.02). Here we show that treatment with the PARP-1 inhibitor veliparib and busulfan elicits a synergistic cytotoxicity in MPN cells both in-vitro and in-vivo. Our data provide the rationale for testing novel pre-transplant conditioning regimens by combining veliparib with alkylating agents, such as busulfan or melphalan, in HSCT for high risk MPNs or MPN-AML. Disclosures Patel: Janssen: Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

scholarly journals Genetic Control of Oxidative Mutagenesis in Sulfolobus acidocaldarius

2020 ◽  
Vol 202 (16) ◽  
Author(s):  
Rupal Jain ◽  
Samuel Dhiman ◽  
Dennis W. Grogan
Keyword(s):  
Dna Polymerase ◽  
Genome Stability ◽  
Dna Oxidation ◽  
Sulfolobus Acidocaldarius ◽  
Hyperthermophilic Archaea ◽  
Genome Replication ◽  
Content Type ◽  
Cellular Survival ◽  
Genes Encoding

ABSTRACT To identify DNA oxidation defenses of hyperthermophilic archaea, we deleted genes encoding the putative 7,8-dihydro-8-oxoguanine (oxoG)-targeted N-glycosylase of Sulfolobus acidocaldarius (ogg; Saci_01367), the Y family DNA polymerase (dbh; Saci_0554), or both and measured the effects on cellular survival, replication accuracy, and oxoG bypass in vivo. Spontaneous G·C-to-T·A transversions were elevated in all Δogg and Δdbh constructs, and the Δogg Δdbh double mutant lost viability at a higher rate than isogenic wild-type (WT) and ogg strains. The distribution of G·C-to-T·A transversions within mutation detector genes suggested that reactivity of G toward oxidation and the effect on translation contribute heavily to the pattern of mutations that are recovered. An impact of the Ogg protein on the overall efficiency of bypassing oxoG in transforming DNA was evident only in the absence of Dbh, and Ogg status did not affect the accuracy of bypass. Dbh function, in contrast, dramatically influenced both the efficiency and accuracy of oxoG bypass. Thus, Ogg and Dbh were found to work independently to avoid mutagenesis by oxoG, and inactivating this simple but effective defense system by deleting both genes imposed a severe mutational burden on S. acidocaldarius cells. IMPORTANCE Hyperthermophilic archaea are expected to have effective (and perhaps atypical) mechanisms to limit the genetic consequences of DNA damage, but few gene products have been demonstrated to have genome-preserving functions in vivo. This study confirmed by genetic criteria that the S. acidocaldarius Ogg protein avoids the characteristic mutagenesis of G oxidation. This enzyme and the bypass polymerase Dbh have similar impacts on genome stability but work independently and may comprise most of the DNA oxidation defense of S. acidocaldarius. The critical dependence of accurate oxoG bypass on the accessory DNA polymerase Dbh further argues that some form of polymerase exchange is important for accurate genome replication in Sulfolobus, and perhaps in related hyperthermophilic archaea.

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