Replicates
Dotaz
Zobrazit nápovědu
1st ed. XXII, 493 s. : fot., obr., rejstř., přeruš.lit. ; 26 cm
Current topics in microbiology and immunology ; 157
259 s. : tab. ; 24 cm
UCLA symposia on molecular and cellular biology ; New series //Vol. 47
782 s. : il.
- MeSH
- rekombinace genetická MeSH
- replikace DNA MeSH
- Publikační typ
- kongresy MeSH
- Konspekt
- Biologické vědy
- NLK Obory
- biologie
- embryologie a teratologie
- genetika, lékařská genetika
In mammalian cells, active ribosomal genes produce the 18S, 5.8S and 28S RNAs of ribosomal particles. Transcription levels of these genes are very high throughout interphase, and the cell needs a special strategy to avoid collision of the DNA polymerase and RNA polymerase machineries. To investigate this problem, we measured the correlation of various replication and transcription signals in the nucleoli of HeLa, HT-1080 and NIH 3T3 cells using a specially devised software for analysis of confocal images. Additionally, to follow the relationship between nucleolar replication and transcription in living cells, we produced a stable cell line expressing GFP-RPA43 (subunit of RNA polymerase I, pol I) and RFP-PCNA (the sliding clamp protein) based on human fibrosarcoma HT-1080 cells. We found that replication and transcription signals are more efficiently separated in nucleoli than in the nucleoplasm. In the course of S phase, separation of PCNA and pol I signals gradually increased. During the same period, separation of pol I and incorporated Cy5-dUTP signals decreased. Analysis of single molecule localization microscopy (SMLM) images indicated that transcriptionally active FC/DFC units (i.e. fibrillar centers with adjacent dense fibrillar components) did not incorporate DNA nucleotides. Taken together, our data show that replication of the ribosomal genes is spatially separated from their transcription, and FC/DFC units may provide a structural basis for that separation.
- MeSH
- buněčné jadérko genetika metabolismus MeSH
- buněčné linie MeSH
- genetická transkripce * MeSH
- HeLa buňky MeSH
- lidé MeSH
- replikace DNA * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The budding yeast Srs2 protein possesses 3' to 5' DNA helicase activity and channels untimely recombination to post-replication repair by removing Rad51 from ssDNA. However, it also promotes recombination via a synthesis-dependent strand-annealing pathway (SDSA). Furthermore, at the replication fork, Srs2 is required for fork progression and prevents the instability of trinucleotide repeats. To better understand the multiple roles of the Srs2 helicase during these processes, we analysed the ability of Srs2 to bind and unwind various DNA substrates that mimic structures present during DNA replication and recombination. While leading or lagging strands were efficiently unwound, the presence of ssDNA binding protein RPA presented an obstacle for Srs2 translocation. We also tested the preferred directionality of unwinding of various substrates and studied the effect of Rad51 and Mre11 proteins on Srs2 helicase activity. These biochemical results help us understand the possible role of Srs2 in the processing of stalled or blocked replication forks as a part of post-replication repair as well as homologous recombination (HR).
- MeSH
- delece genu MeSH
- DNA-helikasy genetika metabolismus MeSH
- endodeoxyribonukleasy metabolismus MeSH
- exodeoxyribonukleasy metabolismus MeSH
- homologní rekombinace * MeSH
- jednovláknová DNA chemie metabolismus MeSH
- křížová struktura DNA chemie metabolismus MeSH
- rekombinasa Rad51 metabolismus MeSH
- replikace DNA * MeSH
- replikační protein A metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus 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.
