Replication Fork Stalling at Natural Impediments

SUMMARY Accurate and complete replication of the genome in every cell division is a prerequisite of genomic stability. Thus, both prokaryotic and eukaryotic replication forks are extremely precise and robust molecular machines that have evolved to be up to the task. However, it has recently become clear that the replication fork is more of a hurdler than a runner: it must overcome various obstacles present on its way. Such obstacles can be called natural impediments to DNA replication, as opposed to external and genetic factors. Natural impediments to DNA replication are particular DNA binding proteins, unusual secondary structures in DNA, and transcription complexes that occasionally (in eukaryotes) or constantly (in prokaryotes) operate on replicating templates. This review describes the mechanisms and consequences of replication stalling at various natural impediments, with an emphasis on the role of replication stalling in genomic instability.

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

10.1101/122895 ◽

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.

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

Science Advances ◽

10.1126/sciadv.abc0330 ◽

2020 ◽

Vol 6 (38) ◽

pp. eabc0330 ◽

Cited By ~ 1

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.

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Role of HP1β during spermatogenesis and DNA replication

10.1101/2019.12.22.886424 ◽

2019 ◽

Author(s):

Vijaya Charaka ◽

Anjana Tiwari ◽

Raj K Pandita ◽

Clayton R Hunt ◽

Tej K. Pandita

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Germ Cells ◽

Chromatin Condensation ◽

Genomic Stability ◽

Replication Forks ◽

Chromatin Protein ◽

Male Germ Cells ◽

Chromatin Compaction

AbstractMaintaining genomic stability in a continually dividing cell population requires accurate DNA repair, especially in male germ cells. Repair and replication protein access to DNA, however, is complicated by chromatin compaction. The HP1β chromatin protein, encoded by Cbx1, is associated with chromatin condensation but its role in meiosis is not clear. To investigate the role of Cbx1 in male germ cells, we generated testis specific Cbx1 deficient transgenic mice by crossing Cbx1flox/flox (Cbx1f/f) mice with Stra8 Cre+/− mice. Loss of Cbx1 in testes adversely affected sperm maturation and Cbx1 deletion increased seminiferous tubule degeneration and basal level DNA damage., We observed that Cbx1−/− MEF cells displayed reduced resolution of stalled DNA replication forks as well as decreased fork restart, indicating defective DNA synthesis. Taken together, these results suggest that loss of Cbx1 in growing cells leads to DNA replication defects and associated DNA damage that impact cell survival.

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Nuclease dead Cas9 is a programmable roadblock for DNA replication

10.1101/455543 ◽

2018 ◽

Cited By ~ 1

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.

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Replisome bypass of transcription complexes and R-loops

Nucleic Acids Research ◽

10.1093/nar/gkaa741 ◽

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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Nuclease dead Cas9 is a programmable roadblock for DNA replication

Scientific Reports ◽

10.1038/s41598-019-49837-z ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 10

Author(s):

Kelsey S. Whinn ◽

Gurleen Kaur ◽

Jacob S. Lewis ◽

Grant D. Schauer ◽

Stefan H. Mueller ◽

...

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Molecular Machines ◽

Enzymatic Activities ◽

Replication Forks ◽

Dna Substrates ◽

Replication Fork Arrest

Abstract Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9–guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro.

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RNF4 Regulates the BLM Helicase in Recovery From Replication Fork Collapse

Frontiers in Genetics ◽

10.3389/fgene.2021.753535 ◽

2021 ◽

Vol 12 ◽

Author(s):

Nathan Ellis ◽

Jianmei Zhu ◽

Mary K Yagle ◽

Wei-Chih Yang ◽

Jing Huang ◽

...

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Replication Stress ◽

Dna Helicase ◽

Replication Forks ◽

Replication Origins ◽

Dna Double Strand Breaks ◽

Response Factors ◽

Fork Stalling ◽

In Cells

Sumoylation is an important enhancer of responses to DNA replication stress and the SUMO-targeted ubiquitin E3 ligase RNF4 regulates these responses by ubiquitylation of sumoylated DNA damage response factors. The specific targets and functional consequences of RNF4 regulation in response to replication stress, however, have not been fully characterized. Here we demonstrated that RNF4 is required for the restart of DNA replication following prolonged hydroxyurea (HU)-induced replication stress. Contrary to its role in repair of γ-irradiation-induced DNA double-strand breaks (DSBs), our analysis revealed that RNF4 does not significantly impact recognition or repair of replication stress-associated DSBs. Rather, using DNA fiber assays, we found that the firing of new DNA replication origins, which is required for replication restart following prolonged stress, was inhibited in cells depleted of RNF4. We also provided evidence that RNF4 recognizes and ubiquitylates sumoylated Bloom syndrome DNA helicase BLM and thereby promotes its proteosome-mediated turnover at damaged DNA replication forks. Consistent with it being a functionally important RNF4 substrate, co-depletion of BLM rescued defects in the firing of new replication origins observed in cells depleted of RNF4 alone. We concluded that RNF4 acts to remove sumoylated BLM from collapsed DNA replication forks, which is required to facilitate normal resumption of DNA synthesis after prolonged replication fork stalling and collapse.

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Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq

PLoS Biology ◽

10.1371/journal.pbio.3000886 ◽

2021 ◽

Vol 19 (3) ◽

pp. e3000886

Author(s):

Neesha Kara ◽

Felix Krueger ◽

Peter Rugg-Gunn ◽

Jonathan Houseley

Keyword(s):

Dna Replication ◽

Mammalian Cells ◽

Replication Fork ◽

Strand Break ◽

Double Strand Break ◽

Replication Forks ◽

Dna Breaks ◽

Genome Wide ◽

Fork Stalling ◽

End Resection

Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3′ ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3′ ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

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Succinylation at a key residue of FEN1 is involved in the DNA damage response to maintain genome stability

AJP Cell Physiology ◽

10.1152/ajpcell.00137.2020 ◽

2020 ◽

Vol 319 (4) ◽

pp. C657-C666

Author(s):

Rongyi Shi ◽

Yiyi Wang ◽

Ya Gao ◽

Xiaoli Xu ◽

Shuyu Mao ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Replication Fork ◽

Genome Stability ◽

Damage Response ◽

Dna Replication And Repair ◽

Fork Stalling ◽

Stalled Replication Forks ◽

Maintain Genome Stability

Human flap endonuclease 1 (FEN1) is a structure-specific, multifunctional endonuclease essential for DNA replication and repair. Our previous study showed that in response to DNA damage, FEN1 interacts with the PCNA-like Rad9-Rad1-Hus1 complex instead of PCNA to engage in DNA repair activities, such as stalled DNA replication fork repair, and undergoes SUMOylation by SUMO-1. Here, we report that succinylation of FEN1 was stimulated in response to DNA replication fork-stalling agents, such as ultraviolet (UV) irradiation, hydroxyurea, camptothecin, and mitomycin C. K200 is a key succinylation site of FEN1 that is essential for gap endonuclease activity and could be suppressed by methylation and stimulated by phosphorylation to promote SUMO-1 modification. Succinylation at K200 of FEN1 promoted the interaction of FEN1 with the Rad9-Rad1-Hus1 complex to rescue stalled replication forks. Impairment of FEN1 succinylation led to the accumulation of DNA damage and heightened sensitivity to fork-stalling agents. Altogether, our findings suggest an important role of FEN1 succinylation in regulating its roles in DNA replication and repair, thus maintaining genome stability.

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Replication-dependent cytotoxicity and Spartan-mediated repair of trapped PARP1–DNA complexes

Nucleic Acids Research ◽

10.1093/nar/gkab777 ◽

2021 ◽

Vol 49 (18) ◽

pp. 10493-10506

Author(s):

Liton Kumar Saha ◽

Yasuhisa Murai ◽

Sourav Saha ◽

Ukhyun Jo ◽

Masataka Tsuda ◽

...

Keyword(s):

Cell Division ◽

Dna Replication ◽

Replication Fork ◽

Protein Complexes ◽

Translesion Synthesis ◽

S Phase ◽

Dna Complexes ◽

Polymerase Inhibitors ◽

Fork Stalling

Abstract The antitumor activity of poly(ADP-ribose) polymerase inhibitors (PARPis) has been ascribed to PARP trapping, which consists in tight DNA–protein complexes. Here we demonstrate that the cytotoxicity of talazoparib and olaparib results from DNA replication. To elucidate the repair of PARP1–DNA complexes associated with replication in human TK6 and chicken DT40 lymphoblastoid cells, we explored the role of Spartan (SPRTN), a metalloprotease associated with DNA replication, which removes proteins forming DPCs. We find that SPRTN-deficient cells are hypersensitive to talazoparib and olaparib, but not to veliparib, a weak PARP trapper. SPRTN-deficient cells exhibit delayed clearance of trapped PARP1 and increased replication fork stalling upon talazoparib and olaparib treatment. We also show that SPRTN interacts with PARP1 and forms nuclear foci that colocalize with the replicative cell division cycle 45 protein (CDC45) in response to talazoparib. Additionally, SPRTN is deubiquitinated and epistatic with translesion synthesis (TLS) in response to talazoparib. Our results demonstrate that SPRTN is recruited to trapped PARP1 in S-phase to assist in the excision and replication bypass of PARP1–DNA complexes.

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