RIF1 promotes replication fork protection and efficient restart to maintain genome stability

ABSTRACTControl of DNA copy number is essential to maintain genome stability and ensure proper cell and tissue function. In Drosophila polyploid cells, the SNF2-domain-containing SUUR protein inhibits replication fork progression within specific regions of the genome to promote DNA underreplication. While dissecting the function of SUUR’s SNF2 domain, we identified a physical interaction between SUUR and Rif1. Rif1 has many roles in DNA metabolism and regulates the replication timing program. We demonstrate that repression of DNA replication is dependent on Rif1. Rif1 localizes to active replication forks in an SUUR-dependent manner and directly regulates replication fork progression. Importantly, SUUR associates with replication forks in the absence of Rif1, indicating that Rif1 acts downstream of SUUR to inhibit fork progression. Our findings uncover an unrecognized function of the Rif1 protein as a regulator of replication fork progression.

Download Full-text

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.

Download Full-text

Rif1 inhibits replication fork progression and controls DNA copy number in Drosophila

eLife ◽

10.7554/elife.39140 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 14

Author(s):

Alexander Munden ◽

Zhan Rong ◽

Amanda Sun ◽

Rama Gangula ◽

Simon Mallal ◽

...

Keyword(s):

Copy Number ◽

Replication Fork ◽

Genome Stability ◽

Replication Timing ◽

Dependent Manner ◽

Replication Forks ◽

Dna Copy Number ◽

Replication Fork Progression ◽

Suur Protein ◽

Maintain Genome Stability

Control of DNA copy number is essential to maintain genome stability and ensure proper cell and tissue function. In Drosophila polyploid cells, the SNF2-domain-containing SUUR protein inhibits replication fork progression within specific regions of the genome to promote DNA underreplication. While dissecting the function of SUUR’s SNF2 domain, we identified an interaction between SUUR and Rif1. Rif1 has many roles in DNA metabolism and regulates the replication timing program. We demonstrate that repression of DNA replication is dependent on Rif1. Rif1 localizes to active replication forks in a partially SUUR-dependent manner and directly regulates replication fork progression. Importantly, SUUR associates with replication forks in the absence of Rif1, indicating that Rif1 acts downstream of SUUR to inhibit fork progression. Our findings uncover an unrecognized function of the Rif1 protein as a regulator of replication fork progression.

Download Full-text

Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork

10.1101/2019.12.18.880690 ◽

2019 ◽

Cited By ~ 1

Author(s):

Domagoj Baretić ◽

Michael Jenkyn-Bedford ◽

Valentina Aria ◽

Giuseppe Cannone ◽

Mark Skehel ◽

...

Keyword(s):

High Resolution ◽

Replication Fork ◽

Genome Stability ◽

Chromosome Replication ◽

Duplex Dna ◽

Multiple Factors ◽

Strand Separation ◽

Fork Protection Complex ◽

Efficient Replication ◽

Maintain Genome Stability

AbstractThe eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG including Ctf4 and the heterotrimeric fork protection complex (Csm3/Tof1 and Mrc1), that have important roles including aiding normal replication rates and stabilizing stalled forks. How these proteins interface with CMG to execute these functions is poorly understood. Here we present 3-3.5 Å resolution cryo-EM structures comprising CMG, Ctf4, Csm3/Tof1 and Mrc1 at a replication fork. The structures provide high-resolution views of CMG:DNA interactions, revealing the mechanism of strand separation. Furthermore, they illustrate the topology of Mrc1 in the replisome and show Csm3/Tof1 ‘grips’ duplex DNA ahead of CMG via a network of interactions that are important for efficient replication fork pausing. Our work reveals how four highly conserved replisome components collaborate with CMG to facilitate replisome progression and maintain genome stability.

Download Full-text

CRISPR-Associated Primase-Polymerases are implicated in prokaryotic CRISPR-Cas adaptation

Nature Communications ◽

10.1038/s41467-021-23535-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Katerina Zabrady ◽

Matej Zabrady ◽

Peter Kolesar ◽

Arthur W. H. Li ◽

Aidan J. Doherty

Keyword(s):

Dna Repair ◽

Dna Synthesis ◽

Genome Stability ◽

Dna Polymerases ◽

Acquired Immunity ◽

Strand Displacement ◽

Genetic Studies ◽

Type Iiia ◽

Maintain Genome Stability ◽

Cas Proteins

AbstractCRISPR-Cas pathways provide prokaryotes with acquired “immunity” against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.

Download Full-text

Coordinated Degradation of Replisome Components Ensures Genome Stability upon Replication Stress in the Absence of the Replication Fork Protection Complex

PLoS Genetics ◽

10.1371/journal.pgen.1003213 ◽

2013 ◽

Vol 9 (1) ◽

pp. e1003213 ◽

Cited By ~ 19

Author(s):

Laura C. Roseaulin ◽

Chiaki Noguchi ◽

Esteban Martinez ◽

Melissa A. Ziegler ◽

Takashi Toda ◽

...

Keyword(s):

Replication Fork ◽

Genome Stability ◽

Replication Stress ◽

Fork Protection Complex

Download Full-text

XRCC1 Prevents Replication Fork Instability during Misincorporation of the DNA Demethylation Bases 5-Hydroxymethyl-2′-Deoxycytidine and 5-Hydroxymethyl-2′-Deoxyuridine

International Journal of Molecular Sciences ◽

10.3390/ijms23020893 ◽

2022 ◽

Vol 23 (2) ◽

pp. 893

Author(s):

María José Peña-Gómez ◽

Marina Suárez-Pizarro ◽

Iván V. Rosado

Keyword(s):

Genomic Instability ◽

Base Excision Repair ◽

Replication Fork ◽

Genome Stability ◽

Excision Repair ◽

Embryonic Stem ◽

Dna Demethylation ◽

Dna Bases ◽

Stem Cell Pluripotency ◽

Base Excision

Whilst avoidance of chemical modifications of DNA bases is essential to maintain genome stability, during evolution eukaryotic cells have evolved a chemically reversible modification of the cytosine base. These dynamic methylation and demethylation reactions on carbon-5 of cytosine regulate several cellular and developmental processes such as embryonic stem cell pluripotency, cell identity, differentiation or tumourgenesis. Whereas these physiological processes are well characterized, very little is known about the toxicity of these cytosine analogues when they incorporate during replication. Here, we report a role of the base excision repair factor XRCC1 in protecting replication fork upon incorporation of 5-hydroxymethyl-2′-deoxycytosine (5hmC) and its deamination product 5-hydroxymethyl-2′-deoxyuridine (5hmU) during DNA synthesis. In the absence of XRCC1, 5hmC exposure leads to increased genomic instability, replication fork impairment and cell lethality. Moreover, the 5hmC deamination product 5hmU recapitulated the genomic instability phenotypes observed by 5hmC exposure, suggesting that 5hmU accounts for the observed by 5hmC exposure. Remarkably, 5hmC-dependent genomic instability and replication fork impairment seen in Xrcc1−/− cells were exacerbated by the trapping of Parp1 on chromatin, indicating that XRCC1 maintains replication fork stability during processing of 5hmC and 5hmU by the base excision repair pathway. Our findings uncover natural epigenetic DNA bases 5hmC and 5hmU as genotoxic nucleosides that threaten replication dynamics and genome integrity in the absence of XRCC1.

Download Full-text

Structure-Specific Endonucleases and the Resolution of Chromosome Underreplication

Genes ◽

10.3390/genes10030232 ◽

2019 ◽

Vol 10 (3) ◽

pp. 232 ◽

Cited By ~ 10

Author(s):

Benoît Falquet ◽

Ulrich Rass

Keyword(s):

Sister Chromatid ◽

Replication Fork ◽

Genome Stability ◽

Molecular Mechanisms ◽

Chromosome Breakage ◽

Repair Synthesis ◽

Daughter Cells ◽

Oncogene Activation ◽

Dna Repair Synthesis ◽

Replication Intermediates

Complete genome duplication in every cell cycle is fundamental for genome stability and cell survival. However, chromosome replication is frequently challenged by obstacles that impede DNA replication fork (RF) progression, which subsequently causes replication stress (RS). Cells have evolved pathways of RF protection and restart that mitigate the consequences of RS and promote the completion of DNA synthesis prior to mitotic chromosome segregation. If there is entry into mitosis with underreplicated chromosomes, this results in sister-chromatid entanglements, chromosome breakage and rearrangements and aneuploidy in daughter cells. Here, we focus on the resolution of persistent replication intermediates by the structure-specific endonucleases (SSEs) MUS81, SLX1-SLX4 and GEN1. Their actions and a recently discovered pathway of mitotic DNA repair synthesis have emerged as important facilitators of replication completion and sister chromatid detachment in mitosis. As RS is induced by oncogene activation and is a common feature of cancer cells, any advances in our understanding of the molecular mechanisms related to chromosome underreplication have important biomedical implications.

Download Full-text

Tim–Tipin dysfunction creates an indispensible reliance on the ATR–Chk1 pathway for continued DNA synthesis

The Journal of Cell Biology ◽

10.1083/jcb.200905006 ◽

2009 ◽

Vol 187 (1) ◽

pp. 15-23 ◽

Cited By ~ 52

Author(s):

Kevin D. Smith ◽

Michael A. Fu ◽

Eric J. Brown

Keyword(s):

Dna Synthesis ◽

Genome Stability ◽

Double Strand Breaks ◽

Dna Unwinding ◽

Replication Forks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Nucleotide Incorporation ◽

Pathway Activation ◽

Maintain Genome Stability

The Tim (Timeless)–Tipin complex has been proposed to maintain genome stability by facilitating ATR-mediated Chk1 activation. However, as a replisome component, Tim–Tipin has also been suggested to couple DNA unwinding to synthesis, an activity expected to suppress single-stranded DNA (ssDNA) accumulation and limit ATR–Chk1 pathway engagement. We now demonstrate that Tim–Tipin depletion is sufficient to increase ssDNA accumulation at replication forks and stimulate ATR activity during otherwise unperturbed DNA replication. Notably, suppression of the ATR–Chk1 pathway in Tim–Tipin-deficient cells completely abrogates nucleotide incorporation in S phase, indicating that the ATR-dependent response to Tim–Tipin depletion is indispensible for continued DNA synthesis. Replication failure in ATR/Tim-deficient cells is strongly associated with synergistic increases in H2AX phosphorylation and DNA double-strand breaks, suggesting that ATR pathway activation preserves fork stability in instances of Tim–Tipin dysfunction. Together, these experiments indicate that the Tim–Tipin complex stabilizes replication forks both by preventing the accumulation of ssDNA upstream of ATR–Chk1 function and by facilitating phosphorylation of Chk1 by ATR.

Download Full-text