scholarly journals RPA phosphorylation regulates DNA resection

2019 ◽  
Author(s):  
Michael M. Soniat ◽  
Logan R. Myler ◽  
Tanya T. Paull ◽  
Ilya J. Finkelstein
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Feedback Loop ◽  
Genetic Recombination ◽  
General Mechanism ◽  
Repair Enzymes ◽  
Blm Helicase ◽  
Damage Response ◽  
Dna Ends ◽  
Dna Resection

AbstractGenetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded (ss) DNA overhangs. Resection termination in bacteria is programmed by a DNA sequence but the mechanisms limiting resection in eukaryotes have remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify a general mechanism that limits DNA resection. BLM helicase together with EXO1 and DNA2 nucleases catalyze kilobase-length DNA resection on nucleosome-coated DNA. The resulting ssDNA is rapidly bound by RPA, which is in turn phosphorylated as part of the DNA damage response (DDR). Remarkably, phosphorylated RPA (pRPA) inhibits DNA resection via regulation of BLM helicase. pRPA suppresses BLM initiation at DNA ends and promotes the intrinsic helicase strand-switching activity. These findings establish that pRPA is a critical regulator of DNA repair enzymes and provides a feedback loop between the DDR and DNA resection termination.

scholarly journals RecG and UvsW catalyse robust DNA rewinding critical for stalled DNA replication fork rescue

2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Maria Manosas ◽  
Senthil K. Perumal ◽  
Piero R. Bianco ◽  
Felix Ritort ◽  
Stephen J. Benkovic ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Genetic Recombination ◽  
Bacteriophage T4 ◽  
General Mechanism ◽  
Dna Unwinding ◽  
Replication Forks ◽  
Displacement Reactions

Abstract Helicases that both unwind and rewind DNA have central roles in DNA repair and genetic recombination. In contrast to unwinding, DNA rewinding by helicases has proved difficult to characterize biochemically because of its thermodynamically downhill nature. Here we use single-molecule assays to mechanically destabilize a DNA molecule and follow, in real time, unwinding and rewinding by two DNA repair helicases, bacteriophage T4 UvsW and Escherichia coli RecG. We find that both enzymes are robust rewinding enzymes, which can work against opposing forces as large as 35 pN, revealing their active character. The generation of work during the rewinding reaction allows them to couple rewinding to DNA unwinding and/or protein displacement reactions central to the rescue of stalled DNA replication forks. The overall results support a general mechanism for monomeric rewinding enzymes.

scholarly journals Mechanistic insights into Lhr helicase function in DNA repair

2020 ◽  
Vol 477 (16) ◽  
pp. 2935-2947
Author(s):  
Ryan J. Buckley ◽  
Kevin Kramm ◽  
Christopher D. O. Cooper ◽  
Dina Grohmann ◽  
Edward L. Bolt
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Single Molecule ◽  
Dna Helicase ◽  
Holliday Junctions ◽  
Single Molecule Fret ◽  
Repair Enzymes ◽  
Dna Strands

The DNA helicase Large helicase-related (Lhr) is present throughout archaea, including in the Asgard and Nanoarchaea, and has homologues in bacteria and eukaryotes. It is thought to function in DNA repair but in a context that is not known. Our data show that archaeal Lhr preferentially targets DNA replication fork structures. In a genetic assay, expression of archaeal Lhr gave a phenotype identical to the replication-coupled DNA repair enzymes Hel308 and RecQ. Purified archaeal Lhr preferentially unwound model forked DNA substrates compared with DNA duplexes, flaps and Holliday junctions, and unwound them with directionality. Single-molecule FRET measurements showed that binding of Lhr to a DNA fork causes ATP-independent distortion and base-pair melting at, or close to, the fork branchpoint. ATP-dependent directional translocation of Lhr resulted in fork DNA unwinding through the ‘parental’ DNA strands. Interaction of Lhr with replication forks in vivo and in vitro suggests that it contributes to DNA repair at stalled or broken DNA replication.

scholarly journals Rolling circle amplification-driven encoding of different fluorescent molecules for simultaneous detection of multiple DNA repair enzymes at the single-molecule level

2020 ◽  
Vol 11 (22) ◽  
pp. 5724-5734
Author(s):  
Chen-chen Li ◽  
Hui-yan Chen ◽  
Juan Hu ◽  
Chun-yang Zhang
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Rolling Circle Amplification ◽  
Simultaneous Detection ◽  
Single Molecule Detection ◽  
Dna Repair Enzymes ◽  
Rolling Circle ◽  
Single Molecule Level ◽  
Repair Enzymes ◽  
Fluorescent Molecules

Integration of single-molecule detection with rolling circle amplification-driven encoding of different fluorescent molecules enables simultaneous detection of multiple DNA repair enzymes.

scholarly journals The Time for Chronotherapy in Radiation Oncology

2021 ◽  
Vol 11 ◽  
Author(s):  
Luis Bermúdez-Guzmán ◽  
Alejandro Blanco-Saborío ◽  
Juliana Ramírez-Zamora ◽  
Eduardo Lovo
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Therapeutic Potential ◽  
Working Hours ◽  
Experimental Models ◽  
Time Range ◽  
Repair Enzymes ◽  
Dna Damage Checkpoints ◽  
Damage Response ◽  
Treatment Administration

Five decades ago, Franz Halberg conceived the idea of ​​a circadian-based therapy for cancer, given the differential tolerance to treatment derived from the intrinsic host rhythms. Nowadays, different experimental models have demonstrated that both the toxicity and efficacy of several anticancer drugs vary by more than 50% as a function of dosing time. Accordingly, it has been shown that chemotherapeutic regimens optimally timed with the circadian cycle have jointly improved patient outcomes both at the preclinical and clinical levels. Along with chemotherapy, radiation therapy is widely used for cancer treatment, but its effectiveness relies mainly on its ability to damage DNA. Notably, the DNA damage response including DNA repair, DNA damage checkpoints, and apoptosis is gated by the circadian clock. Thus, the therapeutic potential of circadian-based radiotherapy against cancer is mainly dependent upon the control that the molecular clock exerts on DNA repair enzymes across the cell cycle. Unfortunately, the time of treatment administration is not usually considered in clinical practice as it varies along the daytime working hours. Currently, only a few studies have evaluated whether the timing of radiotherapy affects the treatment outcome. Several of these studies show that it is possible to reduce the toxicity of the treatment if it is applied at a specific time range, although with some inconsistencies. In this Perspective, we review the main advances in the field of chronoradiotherapy, the possible causes of the inconsistencies observed in the studies so far and provide some recommendations for future trials.

EFFECT OF MMSC AND HSC ON REPARATIVE REGENERATION OF THE LIVER UNDER CONDITIONS OF ITS TOXIC DAMAGE

2020 ◽  
Vol 17 (3) ◽  
pp. 243-248
Author(s):  
I.Yu. Maklakova ◽  
◽  
V.V. Bazarniy ◽  
D.Yu. Grebnev ◽  
◽  
...  
Keyword(s):  
Dna Repair ◽  
Carbon Tetrachloride ◽  
Mitotic Activity ◽  
Liver Damage ◽  
Inflammatory Activity ◽  
Control Group ◽  
Nacl Solution ◽  
Repair Enzymes ◽  
Toxic Liver Damage ◽  
Hsc Transplantation

The aim of this study was to study the effect of combined MMSC and HSC transplantation on liver regeneration under conditions of toxic carbon tetrachloride damage. Materials and methods. The study was performed on white male mice with toxic liver damage by intraperitoneal administration of carbon tetrachloride at a dose of 50 µl per mouse once. An hour after modeling liver damage, placental MMSCs and HSCs were administered intravenously at a dose of 4 million cells/kg and 330 thousand cells/kg, respectively, suspended in 0.2 ml of 0.9% NaCl solution. Control group animals were given 0.9% NaCl solution-0.2 ml intravenously. On days 1, 3, and 7 after cell transplantation, changes in inflammatory activity in the liver were evaluated, and mitotic and apoptotic indices were determined. On the 7th day after the introduction of cells, the activity of DNA repair enzymes of the PARP family was analyzed. Results. Combined MMSC and HSC transplantation leads to a decrease in the index of inflammatory activity in the liver due to a decrease in necrosis, hepatocyte dystrophy, and a decrease in infiltration. As a result of the study, an increase in the activity of PARP repair enzymes was found, which led to a decrease in programmed cell death. Also, cotransplantation of MMSCs and HSCs was accompanied by increased mitotic activity of hepatocytes. Conclusion. Cotransplantation of MMSCs and HSCs under conditions of toxic liver damage reduces the inflammatory response, stimulates the mitotic activity of hepatocytes, and increases the activity of enzymes of the DNA repair system. Activation of the liver's reparative system, in turn, reduces the programmed death of hepatocytes.

scholarly journals Transcriptome analysis and molecular mechanism of linseed (Linum usitatissimum L.) drought tolerance under repeated drought using single-molecule long-read sequencing

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Wei Wang ◽  
Lei Wang ◽  
Ling Wang ◽  
Meilian Tan ◽  
Collins O. Ogutu ◽  
...  
Keyword(s):  
Dna Repair ◽  
Drought Tolerance ◽  
Single Molecule ◽  
Linum Usitatissimum ◽  
Expression Patterns ◽  
Transcriptional Response ◽  
Enrichment Analysis ◽  
Resistant Variety ◽  
Proline Biosynthesis ◽  
Interacting Protein

Abstract Background Oil flax (linseed, Linum usitatissimum L.) is one of the most important oil crops., However, the increases in drought resulting from climate change have dramatically reduces linseed yield and quality, but very little is known about how linseed coordinates the expression of drought resistance gene in response to different level of drought stress (DS) on the genome-wide level. Results To explore the linseed transcriptional response of DS and repeated drought (RD) stress, we determined the drought tolerance of different linseed varieties. Then we performed full-length transcriptome sequencing of drought-resistant variety (Z141) and drought-sensitive variety (NY-17) under DS and RD stress at the seedling stage using single-molecule real-time sequencing and RNA-sequencing. Gene Ontology (GO) and reduce and visualize GO (REVIGO) enrichment analysis showed that upregulated genes of Z141 were enriched in more functional pathways related to plant drought tolerance than those of NY-17 were under DS. In addition, 4436 linseed transcription factors were identified, and 1190 were responsive to stress treatments. Moreover, protein-protein interaction (PPI) network analysis showed that the proline biosynthesis pathway interacts with stress response genes through RAD50 (DNA repair protein 50) interacting protein 1 (RIN-1). Finally, proline biosynthesis and DNA repair structural gene expression patterns were verified by RT- PCR. Conclusions The drought tolerance of Z141 may be related to its upregulation of drought tolerance genes under DS. Proline may play an important role in linseed drought tolerance by maintaining cell osmotic and protecting DNA from ROS damage. In summary, this study provides a new perspective to understand the drought adaptability of linseed.

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