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

2019 ◽  
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.

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

PLoS Genetics ◽  
2013 ◽  
Vol 9 (1) ◽  
pp. e1003213 ◽  
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

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

2018 ◽  
Author(s):  
Alexander Munden ◽  
Zhan Rong ◽  
Rama Gangula ◽  
Simon Mallal ◽  
Jared T. Nordman
Keyword(s):  
Copy Number ◽  
Replication Fork ◽  
Genome Stability ◽  
Replication Timing ◽  
Physical Interaction ◽  
Dependent Manner ◽  
Replication Forks ◽  
Dna Copy Number ◽  
Replication Fork Progression ◽  
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.

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

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Chirantani Mukherjee ◽  
Vivek Tripathi ◽  
Eleni Maria Manolika ◽  
Anne Margriet Heijink ◽  
Giulia Ricci ◽  
...  
Keyword(s):  
Replication Fork ◽  
Genome Stability ◽  
Maintain Genome Stability

scholarly journals SDE2 Integrates into the TIMELESS-TIPIN Complex to Protect Stalled Replication Forks

2020 ◽  
Author(s):  
Julie Rageul ◽  
Jennifer J. Park ◽  
Ping Ping Zeng ◽  
Eun-A Lee ◽  
Jihyeon Yang ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Stability ◽  
Essential Role ◽  
Replication Stress ◽  
Replication Forks ◽  
Genomic Integrity ◽  
Fork Protection Complex ◽  
Stalled Replication Forks ◽  
Stalled Fork

ABSTRACTProtecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances TIM stability and its localization to replication forks, thereby aiding the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

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

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.

scholarly journals DNA unwinding mechanism of a eukaryotic replicative CMG helicase

2019 ◽  
Author(s):  
Zuanning Yuan ◽  
Roxana Georgescu ◽  
Lin Bai ◽  
Dan Zhang ◽  
Huilin Li ◽  
...  
Keyword(s):  
High Resolution ◽  
Replication Fork ◽  
Atomic Resolution ◽  
Dna Unwinding ◽  
Diversion Tunnel ◽  
Strand Separation ◽  
Hairpin Loops ◽  
Ob Fold ◽  
Leading Strand ◽  
Lagging Strand

ABSTRACTHigh-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understand the unwinding mechanism. The eukaryotic replicative CMG helicase contains a Mcm2-7 motor ring, with the N-tier ring in front and the C-tier motor ring behind. The N-tier ring is structurally divided into a zinc finger (ZF) sub-ring followed by the OB fold ring. Here we report the cryo-EM structure of CMG on forked DNA at 3.9 Å, revealing that parental DNA enters the ZF sub-ring and strand separation occurs at the bottom of the ZF sub-ring, where the lagging strand is blocked and diverted sideways by OB hairpin-loops of Mcm3, Mcm4, Mcm6, and Mcm7. Thus, instead of employing a separation pin, unwinding is achieved via a “dam-and-diversion tunnel” for steric exclusion unwinding. The C-tier motor ring contains spirally configured PS1 and H2I loops of Mcms 2, 3, 5, 6 that translocate on the spirally-configured leading strand, and thereby pull the preceding DNA segment through the diversion tunnel for strand separation.

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

eLife ◽  
2018 ◽  
Vol 7 ◽  
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.

Structure of HIRAN domain of human HLTF bound to duplex DNA provides structural basis for DNA unwinding to initiate replication fork regression

2020 ◽  
Vol 167 (6) ◽  
pp. 597-602
Author(s):  
Asami Hishiki ◽  
Mamoru Sato ◽  
Hiroshi Hashimoto
Keyword(s):  
Hydroxy Group ◽  
Replication Fork ◽  
Dna Lesions ◽  
Structural Basis ◽  
Duplex Dna ◽  
Double Stranded Dna ◽  
Strand Separation ◽  
Fork Regression ◽  
Stalled Fork ◽  
Dsdna Binding

Abstract Replication fork regression is a mechanism to rescue a stalled fork by various replication stresses, such as DNA lesions. Helicase-like transcription factor, a SNF2 translocase, plays a central role in the fork regression and its N-terminal domain, HIRAN (HIP116 and Rad5 N-terminal), binds the 3’-hydroxy group of single-stranded DNA. Furthermore, HIRAN is supposed to bind double-stranded DNA (dsDNA) and involved in strand separation in the fork regression, whereas structural basis for mechanisms underlying dsDNA binding and strand separation by HIRAN are still unclear. Here, we report the crystal structure of HIRAN bound to duplex DNA. The structure reveals that HIRAN binds the 3’-hydroxy group of DNA and unexpectedly unwinds three nucleobases of the duplex. Phe-142 is involved in the dsDNA binding and the strand separation. In addition, the structure unravels the mechanism underlying sequence-independent recognition for purine bases by HIRAN, where the N-glycosidic bond adopts syn conformation. Our findings indicate direct involvement of HIRAN in the fork regression by separating of the daughter strand from the parental template.

scholarly journals SDE2 integrates into the TIMELESS-TIPIN complex to protect stalled replication forks

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Julie Rageul ◽  
Jennifer J. Park ◽  
Ping Ping Zeng ◽  
Eun-A Lee ◽  
Jihyeon Yang ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Stability ◽  
Essential Role ◽  
Replication Stress ◽  
Replication Forks ◽  
Genomic Integrity ◽  
Fork Protection Complex ◽  
Stalled Replication Forks ◽  
Stalled Fork

Abstract Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances its stability, thereby aiding TIM localization to replication forks and the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

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

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.

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