scholarly journals Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication

2020 ◽  
Vol 6 (38) ◽  
pp. eabc0330 ◽  
Author(s):  
D. T. Gruszka ◽  
S. Xie ◽  
H. Kimura ◽  
H. Yardimci
Keyword(s):  
Dna Replication ◽  
Real Time ◽  
Single Molecule ◽  
Replication Fork ◽  
Epigenetic Inheritance ◽  
Replication Forks ◽  
Replication Fork Progression ◽  
Egg Extracts ◽  
Fork Stalling ◽  
The Moment

During replication, nucleosomes are disrupted ahead of the replication fork, followed by their reassembly on daughter strands from the pool of recycled parental and new histones. However, because no previous studies have managed to capture the moment that replication forks encounter nucleosomes, the mechanism of recycling has remained unclear. Here, through real-time single-molecule visualization of replication fork progression in Xenopus egg extracts, we determine explicitly the outcome of fork collisions with nucleosomes. Most of the parental histones are evicted from the DNA, with histone recycling, nucleosome sliding, and replication fork stalling also occurring but at lower frequencies. Critically, we find that local histone recycling becomes dominant upon depletion of endogenous histones from extracts, revealing that free histone concentration is a key modulator of parental histone dynamics at the replication fork. The mechanistic details revealed by these studies have major implications for our understanding of epigenetic inheritance.

scholarly journals Replication Fork Slowing and Stalling are Distinct, Checkpoint-Independent Consequences of Replicating Damaged DNA

2017 ◽  
Author(s):  
Divya Ramalingam Iyer ◽  
Nicholas Rhind
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Fission Yeast ◽  
Single Molecule ◽  
Replication Fork ◽  
Replication Forks ◽  
Origin Firing ◽  
Dna Combing ◽  
Fork Stalling

AbstractIn response to DNA damage during S phase, cells slow DNA replication. This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. Slowing of replication allows for tolerance of DNA damage and suppresses genomic instability. Although the mechanisms of origin inhibition by the intra-S checkpoint are understood, major questions remain about how the checkpoint regulates replication forks: Does the checkpoint regulate the rate of fork progression? Does the checkpoint affect all forks, or only those encountering damage? Does the checkpoint facilitate the replication of polymerase-blocking lesions? To address these questions, we have analyzed the checkpoint in the fission yeast Schizosaccharomyces pombe using a single-molecule DNA combing assay, which allows us to unambiguously separate the contribution of origin and fork regulation towards replication slowing, and allows us to investigate the behavior of individual forks. Moreover, we have interrogated the role of forks interacting with individual sites of damage by using three damaging agents—MMS, 4NQO and bleomycin—that cause similar levels of replication slowing with very different frequency of DNA lesions. We find that the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. Finally, using a novel analytic approach, we rigorously identify fork stalling events in our combing data and show that they play a previously unappreciated role in shaping replication kinetics in response to DNA damage.Author SummaryFaithful duplication of the genome is essential for genetic stability of organisms and species. To ensure faithful duplication, cells must be able to replicate damaged DNA. To do so, they employ checkpoints that regulate replication in response to DNA damage. However, the mechanisms by which checkpoints regulate DNA replication forks, the macromolecular machines that contain the helicases and polymerases required to unwind and copy the parental DNA, is unknown. We have used DNA combing, a single-molecule technique that allows us to monitor the progression of individual replication forks, to characterize the response of fission yeast replication forks to DNA damage that blocks the replicative polymerases. We find that forks pass most lesions with only a brief pause and that this lesion bypass is checkpoint independent. However, at a low frequency, forks stall at lesions, and that the checkpoint is required to prevent these stalls from accumulating single-stranded DNA. Our results suggest that the major role of the checkpoint is not to regulate the interaction of replication forks with DNA damage, per se, but to mitigate the consequences of fork stalling when forks are unable to successfully navigate DNA damage on their own.

scholarly journals Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms

2021 ◽  
Author(s):  
Sabrina X. Van Ravenstein ◽  
Kavi P. Mehta ◽  
Tamar Kavlashvili ◽  
Jo Ann Byl ◽  
Runxiang Zhao ◽  
...  
Keyword(s):  
Dna Replication ◽  
Topoisomerase Ii ◽  
Dependent Manner ◽  
Replication Forks ◽  
Dna Breaks ◽  
Replication Fork Progression ◽  
Topoisomerase Ii Poisons ◽  
Egg Extracts ◽  
Fork Stalling ◽  
Major Target

AbstractTopoisomerase II (Top2) unlinks chromosomes during vertebrate DNA replication. Top2 ‘poisons’ are widely-used chemotherapeutics that stabilize Top2 complexes on DNA, leading to cytotoxic DNA breaks. However, it is unclear how these drugs affect DNA replication, which is a major target of Top2 poisons. Using Xenopus egg extracts, we show that the Top2 poisons etoposide and doxorubicin both inhibit DNA replication through different mechanisms. Etoposide induces Top2-dependent DNA breaks and induces Top2-dependent fork stalling by trapping Top2 behind replication forks. In contrast, doxorubicin does not lead to appreciable break formation and instead intercalates into parental DNA to inhibit replication fork progression. In human cells, etoposide stalls replication forks in a Top2-dependent manner, while doxorubicin stalls forks independently of Top2. However, both drugs exhibit Top2-dependent cytotoxicity. Thus, despite shared genetic requirements for cytotoxicity etoposide and doxorubicin inhibit DNA replication through distinct mechanisms.

scholarly journals Mms1 and Mms22 stabilize the replisome during replication stress

2011 ◽  
Vol 22 (13) ◽  
pp. 2396-2408 ◽  
Author(s):  
Jessica A. Vaisica ◽  
Anastasija Baryshnikova ◽  
Michael Costanzo ◽  
Charles Boone ◽  
Grant W. Brown
Keyword(s):  
Dna Replication ◽  
Ubiquitin Ligase ◽  
Replication Fork ◽  
E3 Ubiquitin Ligase ◽  
Replication Stress ◽  
Replication Forks ◽  
Replication Fork Progression ◽  
Binding Partners ◽  
Efficient Recovery ◽  
Stalled Replication Forks

Mms1 and Mms22 form a Cul4Ddb1-like E3 ubiquitin ligase with the cullin Rtt101. In this complex, Rtt101 is bound to the substrate-specific adaptor Mms22 through a linker protein, Mms1. Although the Rtt101Mms1/Mms22ubiquitin ligase is important in promoting replication through damaged templates, how it does so has yet to be determined. Here we show that mms1Δ and mms22Δ cells fail to properly regulate DNA replication fork progression when replication stress is present and are defective in recovery from replication fork stress. Consistent with a role in promoting DNA replication, we find that Mms1 is enriched at sites where replication forks have stalled and that this localization requires the known binding partners of Mms1—Rtt101 and Mms22. Mms1 and Mms22 stabilize the replisome during replication stress, as binding of the fork-pausing complex components Mrc1 and Csm3, and DNA polymerase ε, at stalled replication forks is decreased in mms1Δ and mms22Δ. Taken together, these data indicate that Mms1 and Mms22 are important for maintaining the integrity of the replisome when DNA replication forks are slowed by hydroxyurea and thereby promote efficient recovery from replication stress.

scholarly journals The Human Tim/Tipin Complex Coordinates an Intra-S Checkpoint Response to UV That Slows Replication Fork Displacement

2007 ◽  
Vol 27 (8) ◽  
pp. 3131-3142 ◽  
Author(s):  
Keziban Ünsal-Kaçmaz ◽  
Paul D. Chastain ◽  
Ping-Ping Qu ◽  
Parviz Minoo ◽  
Marila Cordeiro-Stone ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Protein A ◽  
Chain Elongation ◽  
Replication Forks ◽  
Interacting Protein ◽  
Checkpoint Response ◽  
Replication Fork Progression ◽  
Dna Chain ◽  
Interfering Rna

ABSTRACT UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m2 UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.

scholarly journals Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication

2019 ◽  
Author(s):  
Dominika T. Gruszka ◽  
Sherry Xie ◽  
Hiroshi Kimura ◽  
Hasan Yardimci
Keyword(s):  
Cell Division ◽  
Xenopus Laevis ◽  
Single Molecule ◽  
Replication Fork ◽  
Direct Evidence ◽  
Single Molecule Imaging ◽  
Epigenetic Memory ◽  
Close Proximity ◽  
Egg Extracts ◽  
Fork Stalling

SUMMARYFaithful replication of chromatin domains during cell division is fundamental to eukaryotic development. During replication, nucleosomes are disrupted ahead of the replication fork, followed by their rapid reassembly on daughter strands from the pool of recycled parental and newly synthesized histones. Here, we use single-molecule imaging and replication assays in Xenopus laevis egg extracts to determine the outcome of replication fork encounters with nucleosomes. Contrary to current models, the majority of parental histones are evicted from the DNA, with histone recycling, nucleosome sliding and replication fork stalling also occurring but at lower frequencies. The anticipated local histone transfer only becomes dominant upon depletion of free histones from extracts. Our studies provide the first direct evidence that parental histones remain in close proximity to their original locus during recycling and reveal that provision of excess histones results in impaired histone recycling, which has the potential to affect epigenetic memory.

scholarly journals Nuclease dead Cas9 is a programmable roadblock for DNA replication

2018 ◽  
Author(s):  
Kelsey Whinn ◽  
Gurleen Kaur ◽  
Jacob S. Lewis ◽  
Grant Schauer ◽  
Stefan Müller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Molecular Machines ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Single Molecule Level ◽  
Replication Fork Arrest

DNA replication occurs on chromosomal DNA while processes such as DNA repair, recombination and transcription continue. However, we have limited experimental tools to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct fused to the photo-stable dL5 protein fluoromodule as a novel, targetable protein-DNA roadblock for studying replication fork arrest at the single-molecule level in vitro as well as in vivo. We find that the specifically bound dCas9–guideRNA complex arrests viral, bacterial and eukaryotic replication forks in vitro.

scholarly journals Replication Fork Velocities at Adjacent Replication Origins Are Coordinately Modified during DNA Replication in Human Cells

2007 ◽  
Vol 18 (8) ◽  
pp. 3059-3067 ◽  
Author(s):  
Chiara Conti ◽  
Barbara Saccà ◽  
John Herrick ◽  
Claude Lalou ◽  
Yves Pommier ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Spatial Organization ◽  
Genome Duplication ◽  
Functional Organization ◽  
Replication Forks ◽  
Dynamic Regulation ◽  
Timely Completion ◽  
Genome Level

The spatial organization of replicons into clusters is believed to be of critical importance for genome duplication in higher eukaryotes, but its functional organization still remains to be fully clarified. The coordinated activation of origins is insufficient on its own to account for a timely completion of genome duplication when interorigin distances vary significantly and fork velocities are constant. Mechanisms coordinating origin distribution with fork progression are still poorly elucidated, because of technical difficulties of visualizing the process. Taking advantage of a single molecule approach, we delineated and compared the DNA replication kinetics at the genome level in human normal primary and malignant cells. Our results show that replication forks moving from one origin, as well as from neighboring origins, tend to exhibit the same velocity, although the plasticity of the replication program allows for their adaptation to variable interorigin distances. We also found that forks that emanated from closely spaced origins tended to move slower than those associated with long replicons. Taken together, our results indicate a functional role for origin clustering in the dynamic regulation of genome duplication.

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.

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