scholarly journals Beginning at the end: DNA replication within the telomere

2015 ◽  
Vol 210 (2) ◽  
pp. 177-179 ◽  
Author(s):  
Susan A. Gerbi
Keyword(s):  
Dna Replication ◽  
Mouse Chromosome ◽  
Single Molecule ◽  
Template Strand ◽  
Cell Biol ◽  
Chromosome Arm ◽  
Leading Strand ◽  
Wrn Helicase ◽  
Single Molecule Analysis

Using single molecule analysis of replicated DNA (SMARD), Drosopoulos et al. (2015; J. Cell Biol. http://dx.doi.org/10.1083/jcb.201410061) report that DNA replication initiates at measurable frequency within the telomere of mouse chromosome arm 14q. They demonstrate that resolution of G4 structures on the G-rich template strand of the telomere requires some overlapping functions of BLM and WRN helicase for leading strand synthesis.

scholarly journals Replisome bypass of transcription complexes and R-loops

2020 ◽  
Vol 48 (18) ◽  
pp. 10353-10367
Author(s):  
Jan-Gert Brüning ◽  
Kenneth J Marians
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Instability ◽  
Bacterial Dna ◽  
Template Strand ◽  
Replication Fork Progression ◽  
Leading Strand ◽  
Fork Stalling ◽  
Transcription Complexes ◽  
Lagging Strand

Abstract The vast majority of the genome is transcribed by RNA polymerases. G+C-rich regions of the chromosomes and negative superhelicity can promote the invasion of the DNA by RNA to form R-loops, which have been shown to block DNA replication and promote genome instability. However, it is unclear whether the R-loops themselves are sufficient to cause this instability or if additional factors are required. We have investigated replisome collisions with transcription complexes and R-loops using a reconstituted bacterial DNA replication system. RNA polymerase transcription complexes co-directionally oriented with the replication fork were transient blockages, whereas those oriented head-on were severe, stable blockages. On the other hand, replisomes easily bypassed R-loops on either template strand. Replication encounters with R-loops on the leading-strand template (co-directional) resulted in gaps in the nascent leading strand, whereas lagging-strand template R-loops (head-on) had little impact on replication fork progression. We conclude that whereas R-loops alone can act as transient replication blocks, most genome-destabilizing replication fork stalling likely occurs because of proteins bound to the R-loops.

Single molecule analysis of DNA replication

Biochimie ◽  
1999 ◽  
Vol 81 (8-9) ◽  
pp. 859-871 ◽  
Author(s):  
John Herrick ◽  
Aaron Bensimon
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Single Molecule Analysis

scholarly journals BLM helicase facilitates telomere replication during leading strand synthesis of telomeres

2015 ◽  
Vol 210 (2) ◽  
pp. 191-208 ◽  
Author(s):  
William C. Drosopoulos ◽  
Settapong T. Kosiyatrakul ◽  
Carl L. Schildkraut
Keyword(s):  
Single Molecule ◽  
Werner Syndrome ◽  
Telomeric Dna ◽  
G4 Dna ◽  
Genome Wide ◽  
Blm Helicase ◽  
Telomere Replication ◽  
Leading Strand ◽  
Single Molecule Analysis

Based on its in vitro unwinding activity on G-quadruplex (G4) DNA, the Bloom syndrome–associated helicase BLM is proposed to participate in telomere replication by aiding fork progression through G-rich telomeric DNA. Single molecule analysis of replicated DNA (SMARD) was used to determine the contribution of BLM helicase to telomere replication. In BLM-deficient cells, replication forks initiating from origins within the telomere, which copy the G-rich strand by leading strand synthesis, moved slower through the telomere compared with the adjacent subtelomere. Fork progression through the telomere was further slowed in the presence of a G4 stabilizer. Using a G4-specific antibody, we found that deficiency of BLM, or another G4-unwinding helicase, the Werner syndrome-associated helicase WRN, resulted in increased G4 structures in cells. Importantly, deficiency of either helicase led to greater increases in G4 DNA detected in the telomere compared with G4 seen genome-wide. Collectively, our findings are consistent with BLM helicase facilitating telomere replication by resolving G4 structures formed during copying of the G-rich strand by leading strand synthesis.

scholarly journals Single-molecule analysis of DNA replication in Xenopus egg extracts

Methods ◽  
2012 ◽  
Vol 57 (2) ◽  
pp. 179-186 ◽  
Author(s):  
Hasan Yardimci ◽  
Anna B. Loveland ◽  
Antoine M. van Oijen ◽  
Johannes C. Walter
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Single Molecule Analysis

scholarly journals Single molecule analysis of Trypanosoma brucei DNA replication dynamics

2015 ◽  
Vol 43 (5) ◽  
pp. 2655-2665 ◽  
Author(s):  
Simone Guedes Calderano ◽  
William C. Drosopoulos ◽  
Marina Mônaco Quaresma ◽  
Catarina A. Marques ◽  
Settapong Kosiyatrakul ◽  
...  
Keyword(s):  
Dna Replication ◽  
Trypanosoma Brucei ◽  
Single Molecule ◽  
Single Molecule Analysis

scholarly journals Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing

BIO-PROTOCOL ◽  
2017 ◽  
Vol 7 (11) ◽  
Author(s):  
Hélène Tourrière ◽  
Julie Saksouk ◽  
Armelle Lengronne ◽  
Philippe Pasero
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Budding Yeast ◽  
Human Cells ◽  
Dna Combing ◽  
Single Molecule Analysis

scholarly journals Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis

2013 ◽  
Vol 24 (5) ◽  
pp. 578-587 ◽  
Author(s):  
Hui Hua ◽  
Mandana Namdar ◽  
Olivier Ganier ◽  
Juraj Gregan ◽  
Marcel Méchali ◽  
...  
Keyword(s):  
Dna Replication ◽  
Chromosome Segregation ◽  
Single Molecule ◽  
Replication Forks ◽  
Cyclin Dependent Kinase ◽  
Chromatin Binding ◽  
Control Factors ◽  
Spindle Disassembly ◽  
Single Molecule Analysis ◽  
Meiosis I

Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit, in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication, but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin-dependent kinase activity, activation of Dbf4-dependent kinase, or deletion of the Spd1 inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, and thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I–II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry.

scholarly journals Mapping DNA replication with nanopore sequencing

2018 ◽  
Author(s):  
Magali Hennion ◽  
Jean-Michel Arbona ◽  
Corinne Cruaud ◽  
Florence Proux ◽  
Benoît Le Tallec ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Electrical Signal ◽  
Yeast Cells ◽  
Nanopore Sequencing ◽  
Single Molecule Level ◽  
Experimental Systems ◽  
Genome Wide ◽  
Single Molecule Analysis

ABSTRACTWe have harnessed nanopore sequencing to study DNA replication genome-wide at the single-molecule level. Using in vitro prepared DNA substrates, we characterized the effect of bromodeoxyuridine (BrdU) substitution for thymidine on the MinION nanopore electrical signal. Using a neural-network basecaller trained on yeast DNA containing either BrdU or thymidine, we identified BrdU-labelled tracts in yeast cells synchronously entering S phase in the presence of hydroxyurea and BrdU. As expected, the BrdU-labelled tracts coincided with previously identified early-firing, but not late-firing, replication origins. These results open the way to high-throughput, high-resolution, single-molecule analysis of DNA replication in many experimental systems.

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