Okazaki fragment
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Poly(ADP-ribose) is synthesized by PARP enzymes during the repair of stochastic DNA breaks. Surprisingly, however, we show that most if not all endogenous poly(ADP-ribose) is detected in normal S phase cells at sites of DNA replication. This S phase poly(ADP-ribose) does not result from damaged or misincorporated nucleotides or from DNA replication stress. Rather, perturbation of the DNA replication proteins LIG1 or FEN1 increases S phase poly(ADP-ribose) more than 10-fold, implicating unligated Okazaki fragments as the source of S phase PARP activity. Indeed, S phase PARP activity is ablated by suppressing Okazaki fragment formation with emetine, a DNA replication inhibitor that selectively inhibits lagging strand synthesis. Importantly, PARP activation during DNA replication recruits the single-strand break repair protein XRCC1, and human cells lacking PARP activity and/or XRCC1 are hypersensitive to FEN1 perturbation. Collectively, our data indicate that PARP1 is a sensor of unligated Okazaki fragments during DNA replication and facilitates their repair.
- MeSH
- "flap" endonukleasy metabolismus MeSH
- buněčné linie MeSH
- DNA vazebné proteiny metabolismus MeSH
- DNA-ligasa ATP metabolismus MeSH
- DNA genetika metabolismus MeSH
- lidé MeSH
- oprava DNA MeSH
- poly(ADP-ribosa)polymerasa 1 metabolismus MeSH
- poly(ADP-ribosa)polymerasy genetika metabolismus MeSH
- polyadenosindifosfátribosa metabolismus MeSH
- poškození DNA MeSH
- protein XRCC1 metabolismus MeSH
- replikace DNA fyziologie MeSH
- S fáze fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DNA synthesis of the leading and lagging strands works independently and cells tolerate single-stranded DNA generated during strand uncoupling if it is protected by RPA molecules. Natural alkaloid emetine is used as a specific inhibitor of lagging strand synthesis, uncoupling leading and lagging strand replication. Here, by analysis of lagging strand synthesis inhibitors, we show that despite emetine completely inhibiting DNA replication: it does not induce the generation of single-stranded DNA and chromatin-bound RPA32 (CB-RPA32). In line with this, emetine does not activate the replication checkpoint nor DNA damage response. Emetine is also an inhibitor of proteosynthesis and ongoing proteosynthesis is essential for the accurate replication of DNA. Mechanistically, we demonstrate that the acute block of proteosynthesis by emetine temporally precedes its effects on DNA replication. Thus, our results are consistent with the hypothesis that emetine affects DNA replication by proteosynthesis inhibition. Emetine and mild POLA1 inhibition prevent S-phase poly(ADP-ribosyl)ation. Collectively, our study reveals that emetine is not a specific lagging strand synthesis inhibitor with implications for its use in molecular biology.
We present Mass Spectrometry-Data Independent Analysis software version 4 (MS-DIAL 4), a comprehensive lipidome atlas with retention time, collision cross-section and tandem mass spectrometry information. We formulated mass spectral fragmentations of lipids across 117 lipid subclasses and included ion mobility tandem mass spectrometry. Using human, murine, algal and plant biological samples, we annotated and semiquantified 8,051 lipids using MS-DIAL 4 with a 1-2% estimated false discovery rate. MS-DIAL 4 helps standardize lipidomics data and discover lipid pathways.
Poly(ADP-ribose) polymerase 1 (PARP1) is implicated in the detection and processing of unligated Okazaki fragments and other DNA replication intermediates, highlighting such structures as potential sources of genome breakage induced by PARP inhibition. Here, we show that PARP1 activity is greatly elevated in chicken and human S phase cells in which FEN1 nuclease is genetically deleted and is highest behind DNA replication forks. PARP inhibitor reduces the integrity of nascent DNA strands in both wild-type chicken and human cells during DNA replication, and does so in FEN1-/- cells to an even greater extent that can be detected as postreplicative single-strand nicks or gaps. Collectively, these data show that PARP inhibitors impede the maturation of nascent DNA strands during DNA replication, and implicate unligated Okazaki fragments and other nascent strand discontinuities in the cytotoxicity of these compounds.
Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.
- MeSH
- "flap" endonukleasy genetika metabolismus terapeutické užití MeSH
- enzymy opravy DNA genetika MeSH
- exodeoxyribonukleasy genetika MeSH
- glykosidhydrolasy genetika metabolismus MeSH
- lidé MeSH
- nádorový supresorový protein p53 * genetika metabolismus MeSH
- nádory * farmakoterapie genetika MeSH
- oprava DNA MeSH
- PARP inhibitory farmakologie MeSH
- poškození DNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The sliding clamp, PCNA, plays a central role in DNA replication and repair. In the moving replication fork, PCNA is present at the leading strand and at each of the Okazaki fragments that are formed on the lagging strand. PCNA enhances the processivity of the replicative polymerases and provides a landing platform for other proteins and enzymes. The loading of the clamp onto DNA is performed by the Replication Factor C (RFC) complex, whereas its unloading can be carried out by an RFC-like complex containing Elg1. Mutations in ELG1 lead to DNA damage sensitivity and genome instability. To characterize the role of Elg1 in maintaining genomic integrity, we used homology modeling to generate a number of site-specific mutations in ELG1 that exhibit different PCNA unloading capabilities. We show that the sensitivity to DNA damaging agents and hyper-recombination of these alleles correlate with their ability to unload PCNA from the chromatin. Our results indicate that retention of modified and unmodified PCNA on the chromatin causes genomic instability. We also show, using purified proteins, that the Elg1 complex inhibits DNA synthesis by unloading SUMOylated PCNA from the DNA. Additionally, we find that mutations in ELG1 suppress the sensitivity of rad5Δ mutants to DNA damage by allowing trans-lesion synthesis to take place. Taken together, the data indicate that the Elg1-RLC complex plays an important role in the maintenance of genomic stability by unloading PCNA from the chromatin.
- MeSH
- chromatin metabolismus MeSH
- DNA-helikasy genetika MeSH
- DNA biosyntéza MeSH
- methylmethansulfonát toxicita MeSH
- mutace MeSH
- nestabilita genomu * MeSH
- poškození DNA * MeSH
- proliferační antigen buněčného jádra metabolismus MeSH
- rekombinace genetická MeSH
- Saccharomyces cerevisiae - proteiny chemie genetika metabolismus MeSH
- strukturní homologie proteinů MeSH
- suprese genetická MeSH
- transportní proteiny chemie genetika metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
... chromozómu 64 -- 3.1.1 Replikačni počátek je komplexní regulační úsek 65 -- 3.1.2 Iniciace syntézy Okazakiho ... ... fragmentů 67 -- 3.1.3 Regulační aspekty iniciace replikace chromozómu 70 -- 3.2 Regulace replikace mimochromozómových ...
253 s. : tab., grafy ; 24 cm
Kniha představuje monografické zpracování poznatků molekulární genetiky.
- Konspekt
- Mikrobiologie
- NLK Obory
- mikrobiologie, lékařská mikrobiologie
... 111 -- 6: Isolating the gene 115 -- A restriction map is constructed by cleaving DNA into specific fragments ... ... DNA synthesis is semidiscontinuous and primed by RNA 477 -- The primosome initiates synthesis of Okazaki ... ... fragments 480 -- Coordinating synthesis of the lagging and leading strands 484 -- The replication apparatus ...
xviii, 1260 stran : ilustrace ; 28 cm
... Zdvojování genetické paměti (replikace DNA) 133 -- Princip replikace DNA 134 -- DNA polymerázy 135 -- Okazakiho ... ... fragmenty 136 -- Rozvinování molekuly DNA 137 -- Ukončení replikace lineárních DNA molekul, syntéza ...
3. přepr. vyd, 1. v nakl. H & H 554 s. : il. ; 26 cm
Kniha je třetím přepracovaným vydáním oblíbené učebnice určené pro lékařské fakulty. Proti předchozím vydáním je doplněna nejmodernějšími poznatky, především z oblasti buněčné a molekulární biologie, které závažným způsobem mění dosavadní pohled na základní pochody v živých systémech. Kniha je určena především studentům lékařských fakult, může však být studijní pomůckou i pro studenty dalších fakult.
- Konspekt
- Biologické vědy
- NLK Obory
- biologie
- NLK Publikační typ
- učebnice vysokých škol
