scholarly journals The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Charanya Kumar ◽  
Sahil Batra ◽  
Jack D Griffith ◽  
Dirk Remus
Keyword(s):  
Genome Instability ◽  
Hybrid Structure ◽  
Replication Fork Progression ◽  
Rna:Dna Hybrid ◽  
Eukaryotic Dna ◽  
Intrinsic Capacity ◽  
Leading Strand ◽  
Rna:Dna Hybrids ◽  
Mechanistic Basis ◽  
Fork Stalling

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.

scholarly journals The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions

2021 ◽  
Author(s):  
Charanya Kumar ◽  
Sahil Batra ◽  
Jack Griffith ◽  
Dirk Remus
Keyword(s):  
Genome Instability ◽  
Hybrid Structure ◽  
Replication Fork Progression ◽  
Rna:Dna Hybrid ◽  
Eukaryotic Dna ◽  
Intrinsic Capacity ◽  
Leading Strand ◽  
Rna:Dna Hybrids ◽  
Mechanistic Basis ◽  
Fork Stalling

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.

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.

scholarly journals A stable leading strand polymerase/clamp loader complex is required for normal and perturbed eukaryotic DNA replication

2019 ◽  
Author(s):  
Katy Stokes ◽  
Alicja Winczura ◽  
Boyuan Song ◽  
Giacomo De Piccoli ◽  
Daniel B. Grabarczyk
Keyword(s):  
Replication Fork ◽  
Catalytic Domain ◽  
Replication Forks ◽  
Yeast Genetics ◽  
Clamp Loader ◽  
Checkpoint Signaling ◽  
Chromatid Cohesion ◽  
Eukaryotic Dna ◽  
Leading Strand ◽  
Fork Stalling

AbstractThe eukaryotic replisome must faithfully replicate DNA and cope with replication fork blocks and stalling, while simultaneously promoting sister chromatid cohesion. Ctf18-RFC is an alternative PCNA loader that links all these processes together by an unknown mechanism. Here, we use integrative structural biology combined with yeast genetics and biochemistry to highlight the specific functions that Ctf18-RFC plays within the leading strand machinery via an interaction with the catalytic domain of DNA Pol ε. We show that a large and unusually flexible interface enables this interaction to occur constitutively throughout the cell cycle and regardless of whether forks are replicating or stalled. We reveal that, by being anchored to the leading strand polymerase, Ctf18-RFC can rapidly signal fork stalling to activate the S phase checkpoint. Moreover, we demonstrate that, independently of checkpoint signaling or chromosome cohesion, Ctf18-RFC functions in parallel to Chl1 and Mrc1 to protect replication forks and cell viability.

scholarly journals DNA Replication Modulates R-loop Levels to Maintain Genome Stability

2017 ◽  
Author(s):  
Stephan Hamperl ◽  
Joshua Saldivar ◽  
Michael Bocek ◽  
Karlene A. Cimprich
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Genome Stability ◽  
Genome Instability ◽  
Loop Formation ◽  
Origin Firing ◽  
Disease States ◽  
Replication Fork Progression ◽  
Mechanistic Basis ◽  
Transcription And Replication

SummaryConflicts between transcription and replication are a potent source of DNA damage. The transcription machinery has the potential to aggravate such conflicts by generating co-transcriptional R-loops as an additional barrier to replication fork progression. Here, we investigate the influence of conflict orientation and R-loop formation on genome stability in human cells using a defined episomal system. This approach reveals that head-on (HO) and co-directional (CD) conflicts induce distinct DNA damage responses. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the CD orientation but promoting their formation in the HO orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings not only uncover an intrinsic function of the replisome in R-loop homeostasis, but also suggest a mechanistic basis for genome instability associated with deregulated DNA replication, which is observed in many disease states, including cancer.

scholarly journals A concomitant loss of dormant origins and FANCC exacerbates genome instability by impairing DNA replication fork progression

2014 ◽  
Vol 42 (9) ◽  
pp. 5605-5615 ◽  
Author(s):  
Spencer W. Luebben ◽  
Tsuyoshi Kawabata ◽  
Charles S. Johnson ◽  
M. Gerard O'Sullivan ◽  
Naoko Shima
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Instability ◽  
Replication Fork Progression

scholarly journals Host Cell Detection of Noncoding Stuffer DNA Contained in Helper-Dependent Adenovirus Vectors Leads to Epigenetic Repression of Transgene Expression

2009 ◽  
Vol 83 (17) ◽  
pp. 8409-8417 ◽  
Author(s):  
P. Joel Ross ◽  
Michael A. Kennedy ◽  
Robin J. Parks
Keyword(s):  
Transgene Expression ◽  
Great Promise ◽  
Cell Detection ◽  
Nuclear Factors ◽  
Insulator Element ◽  
Pml Bodies ◽  
Eukaryotic Dna ◽  
Foreign Dna ◽  
Mechanistic Basis ◽  
Epigenetic Repression

ABSTRACT Helper-dependent adenovirus (hdAd) vectors have shown great promise as therapeutic gene delivery vehicles in gene therapy applications. However, the level and duration of gene expression from hdAd can differ considerably depending on the nature of the noncoding stuffer DNA contained within the vector. For example, an hdAd containing 22 kb of prokaryotic DNA (hdAd-prok) expresses its transgene 60-fold less efficiently than a similar vector containing eukaryotic DNA (hdAd-euk). Here we have determined the mechanistic basis of this phenomenon. Although neither vector was subjected to CpG methylation and both genomes associated with cellular histones to similar degrees, hdAd-prok chromatin was actively deacetylated. Insertion of an insulator element between the transgene and the bacterial DNA derepressed hdAd-prok, suggesting that foreign DNA nucleates repressive chromatin structures that spread to the transgene. We found that Sp100B/Sp100HMG and Daxx play a role in repressing transgene expression from hdAd and act independently of PML bodies. Thus, we have identified nuclear factors involved in recognizing foreign DNA and have determined the mechanism by which associated genes are repressed.

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 Ctf18-RFC and DNA Pol ϵ form a stable leading strand polymerase/clamp loader complex required for normal and perturbed DNA replication

2020 ◽  
Vol 48 (14) ◽  
pp. 8128-8145 ◽  
Author(s):  
Katy Stokes ◽  
Alicja Winczura ◽  
Boyuan Song ◽  
Giacomo De Piccoli ◽  
Daniel B Grabarczyk
Keyword(s):  
Structural Biology ◽  
Replication Fork ◽  
Catalytic Domain ◽  
Replication Forks ◽  
Yeast Genetics ◽  
Clamp Loader ◽  
Checkpoint Signaling ◽  
Chromatid Cohesion ◽  
Leading Strand ◽  
Fork Stalling

Abstract The eukaryotic replisome must faithfully replicate DNA and cope with replication fork blocks and stalling, while simultaneously promoting sister chromatid cohesion. Ctf18-RFC is an alternative PCNA loader that links all these processes together by an unknown mechanism. Here, we use integrative structural biology combined with yeast genetics and biochemistry to highlight the specific functions that Ctf18-RFC plays within the leading strand machinery via an interaction with the catalytic domain of DNA Pol ϵ. We show that a large and unusually flexible interface enables this interaction to occur constitutively throughout the cell cycle and regardless of whether forks are replicating or stalled. We reveal that, by being anchored to the leading strand polymerase, Ctf18-RFC can rapidly signal fork stalling to activate the S phase checkpoint. Moreover, we demonstrate that, independently of checkpoint signaling or chromosome cohesion, Ctf18-RFC functions in parallel to Chl1 and Mrc1 to protect replication forks and cell viability.

scholarly journals Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
André Franz ◽  
Paul A. Pirson ◽  
Domenic Pilger ◽  
Swagata Halder ◽  
Divya Achuthankutty ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dynamic Process ◽  
Metabolic Pathways ◽  
Replication Fork ◽  
Genome Instability ◽  
Genome Integrity ◽  
Substrate Selection ◽  
Replication Fork Progression ◽  
Replication Factors ◽  
Spatiotemporal Control

Abstract The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging.

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