scholarly journals The Fml1-MHF complex suppresses inter-fork strand annealing in fission yeast

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Io Nam Wong ◽  
Jacqueline PS Neo ◽  
Judith Oehler ◽  
Sophie Schafhauser ◽  
Fekret Osman ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Dna Replication ◽  
Fission Yeast ◽  
Replication Fork ◽  
Dna Helicase ◽  
Genomic Deletions ◽  
Terminal Domain ◽  
Plausible Mechanism ◽  
Strand Annealing

Previously we reported that a process called inter-fork strand annealing (IFSA) causes genomic deletions during the termination of DNA replication when an active replication fork converges on a collapsed fork (Morrow et al., 2017). We also identified the FANCM-related DNA helicase Fml1 as a potential suppressor of IFSA. Here, we confirm that Fml1 does indeed suppress IFSA, and show that this function depends on its catalytic activity and ability to interact with Mhf1-Mhf2 via its C-terminal domain. Finally, a plausible mechanism of IFSA suppression is demonstrated by the finding that Fml1 can catalyse regressed fork restoration in vitro.

scholarly journals Chromatin remodeler Fft3 plays a dual role at blocked DNA replication forks

2019 ◽  
Vol 2 (5) ◽  
pp. e201900433 ◽  
Author(s):  
Anissia Ait-Saada ◽  
Olga Khorosjutina ◽  
Jiang Chen ◽  
Karol Kramarz ◽  
Vladimir Maksimov ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Fission Yeast ◽  
Chromatin Remodeling ◽  
Replication Fork ◽  
Strand Break ◽  
Replication Forks ◽  
Damage Repair ◽  
Single Strand Annealing ◽  
Strand Annealing

Here, we investigate the function of fission yeast Fun30/Smarcad1 family of SNF2 ATPase-dependent chromatin remodeling enzymes in DNA damage repair. There are three Fun30 homologues in fission yeast, Fft1, Fft2, and Fft3. We find that only Fft3 has a function in DNA repair and it is needed for single-strand annealing of an induced double-strand break. Furthermore, we use an inducible replication fork barrier system to show that Fft3 has two distinct roles at blocked DNA replication forks. First, Fft3 is needed for the resection of nascent strands, and second, it is required to restart the blocked forks. The latter function is independent of its ATPase activity.

Prokaryotic DNA replication mechanisms

1987 ◽  
Vol 317 (1187) ◽  
pp. 395-420 ◽  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Kinetic Studies ◽  
Dna Helicase ◽  
Gene 32 ◽  
Dna Template ◽  
Lagging Strand

The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology. In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase). DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity. DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers). Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves. Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.

scholarly journals Two components of DNA replication-dependent LexA cleavage

2020 ◽  
Vol 295 (30) ◽  
pp. 10368-10379
Author(s):  
Kamila K. Myka ◽  
Kenneth J. Marians
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Dna Helicase ◽  
Gene Products ◽  
Cyclobutane Pyrimidine Dimers ◽  
Pyrimidine Dimers ◽  
Cleavage Assay ◽  
Leading Strand ◽  
Cycle Regulation

Induction of the SOS response, a cellular system triggered by DNA damage in bacteria, depends on DNA replication for the generation of the SOS signal, ssDNA. RecA binds to ssDNA, forming filaments that stimulate proteolytic cleavage of the LexA transcriptional repressor, allowing expression of > 40 gene products involved in DNA repair and cell cycle regulation. Here, using a DNA replication system reconstituted in vitro in tandem with a LexA cleavage assay, we studied LexA cleavage during DNA replication of both undamaged and base-damaged templates. Only a ssDNA–RecA filament supported LexA cleavage. Surprisingly, replication of an undamaged template supported levels of LexA cleavage like that induced by a template carrying two site-specific cyclobutane pyrimidine dimers. We found that two processes generate ssDNA that could support LexA cleavage. 1) During unperturbed replication, single-stranded regions formed because of stochastic uncoupling of the leading-strand DNA polymerase from the replication fork DNA helicase, and 2) on the damaged template, nascent leading-strand gaps were generated by replisome lesion skipping. The two pathways differed in that RecF stimulated LexA cleavage during replication of the damaged template, but not normal replication. RecF appears to facilitate RecA filament formation on the leading-strand ssDNA gaps generated by replisome lesion skipping.

scholarly journals Inter-Fork Strand Annealing causes genomic deletions during the termination of DNA replication

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Carl A Morrow ◽  
Michael O Nguyen ◽  
Andrew Fower ◽  
Io Nam Wong ◽  
Fekret Osman ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Sequences ◽  
Genetic Disorders ◽  
Dna Helicase ◽  
Potential Threat ◽  
Genomic Deletions ◽  
Recombination Proteins ◽  
Replication Restart ◽  
Strand Annealing ◽  
Human Genetic Disorders

Problems that arise during DNA replication can drive genomic alterations that are instrumental in the development of cancers and many human genetic disorders. Replication fork barriers are a commonly encountered problem, which can cause fork collapse and act as hotspots for replication termination. Collapsed forks can be rescued by homologous recombination, which restarts replication. However, replication restart is relatively slow and, therefore, replication termination may frequently occur by an active fork converging on a collapsed fork. We find that this type of non-canonical fork convergence in fission yeast is prone to trigger deletions between repetitive DNA sequences via a mechanism we call Inter-Fork Strand Annealing (IFSA) that depends on the recombination proteins Rad52, Exo1 and Mus81, and is countered by the FANCM-related DNA helicase Fml1. Based on our findings, we propose that IFSA is a potential threat to genomic stability in eukaryotes.

scholarly journals DNA Unwinding Is an MCM Complex-dependent and ATP Hydrolysis-dependent Process

2004 ◽  
Vol 279 (44) ◽  
pp. 45586-45593 ◽  
Author(s):  
David Shechter ◽  
Carol Y. Ying ◽  
Jean Gautier
Keyword(s):  
Dna Replication ◽  
Neutralizing Antibodies ◽  
Atp Hydrolysis ◽  
Initial Period ◽  
Dna Helicase ◽  
Dna Unwinding ◽  
Origin Firing ◽  
Replicative Helicase ◽  
Mcm Proteins

Minichromosome maintenance proteins (Mcm) are essential in all eukaryotes and are absolutely required for initiation of DNA replication. The eukaryotic and archaeal Mcm proteins have conserved helicase motifs and exhibit DNA helicase and ATP hydrolysis activitiesin vitro. Although the Mcm proteins have been proposed to be the replicative helicase, the enzyme that melts the DNA helix at the replication fork, their function during cellular DNA replication elongation is still unclear. Using nucleoplasmic extract (NPE) fromXenopus laeviseggs and six purified polyclonal antibodies generated against each of theXenopusMcm proteins, we have demonstrated that Mcm proteins are required during DNA replication and DNA unwinding after initiation of replication. Quantitative depletion of Mcms from the NPE results in normal replication and unwinding, confirming that Mcms are required before pre-replicative complex assembly and dispensable thereafter. Replication and unwinding are inhibited when pooled neutralizing antibodies against the six different Mcm2–7 proteins are added during NPE incubation. Furthermore, replication is blocked by the addition of the Mcm antibodies after an initial period of replication in the NPE, visualized by a pulse of radiolabeled nucleotide at the same time as antibody addition. Addition of the cyclin-dependent kinase 2 inhibitor p21cip1specifically blocks origin firing but does not prevent helicase action. When p21cip1is added, followed by the non-hydrolyzable analog ATPγS to block helicase function, unwinding is inhibited, demonstrating that plasmid unwinding is specifically attributable to an ATP hydrolysis-dependent function. These data support the hypothesis that the Mcm protein complex functions as the replicative helicase.

scholarly journals Threonine-11, Phosphorylated by Rad3 and ATM In Vitro, Is Required for Activation of Fission Yeast Checkpoint Kinase Cds1

2001 ◽  
Vol 21 (10) ◽  
pp. 3398-3404 ◽  
Author(s):  
Katsunori Tanaka ◽  
Michael N. Boddy ◽  
Xiao-Bo Chen ◽  
Clare H. McGowan ◽  
Paul Russell
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Fission Yeast ◽  
Ataxia Telangiectasia ◽  
Null Mutant ◽  
Ataxia Telangiectasia Mutated ◽  
Tolerance Mechanisms ◽  
Checkpoint Kinase ◽  
And Function

ABSTRACT Fission yeast Cds1 is phosphorylated and activated when DNA replication is interrupted by nucleotide starvation or DNA damage. Cds1 enforces the S-M checkpoint that couples mitosis (M) to the completion of DNA synthesis (S). Cds1 also controls replicational stress tolerance mechanisms. Cds1 is regulated by a group of proteins that includes Rad3, a kinase related to human checkpoint kinase ATM (ataxia telangiectasia mutated). ATM phosphorylates serine or threonine followed by glutamine (SQ or TQ). Here we show that in vitro, Rad3 and ATM phosphorylate the N-terminal domain of Cds1 at the motif T11Q12. Substitution of threonine-11 with alanine (T11A) abolished Cds1 activation that occurs when DNA replication is inhibited by hydroxyurea (HU) treatment. Thecds1-T11A mutant was profoundly sensitive to HU, although not quite as sensitive as a cds1− null mutant. Cds1T11A was unable to enforce the S-M checkpoint. These results strongly suggest that Rad3-dependent phosphorylation of Cds1 at threonine-11 is required for Cds1 activation and function.

scholarly journals ALKOXYALKYL ESTERS OF NUCLEOTIDE ANALOGS INHIBIT POLYOMAVIRUS DNA REPLICATION AND LARGE T ANTIGEN ACTIVITIES

2020 ◽  
Author(s):  
Nichodemus O. Onwubiko ◽  
Suraya Diaz ◽  
Marcela Krecmerova ◽  
Heinz Peter Nasheuer
Keyword(s):  
Dna Replication ◽  
Tumor Antigens ◽  
Antiviral Agent ◽  
Dna Helicase ◽  
T Antigen ◽  
Nucleotide Analogs ◽  
Large Tumor ◽  
Nucleotide Analog ◽  
Derivatives Of

Polyomavirus-related infections are ubiqutious in immunocompromised individuals and in some cases are intractable and fatal. Due to lack of approved drugs to treat polyomavirus infections, cidofovir, a phosphonate nucleotide analog approved to treat cytomegalovirus infections has been repurposed as anti-polyomavirus agent. Cidofovir has been modified in various ways to improve its efficacies as broad-spectrum antiviral agent. However, the actual mechanisms and targets of cidofovir and its modified derivatives as anti-polyomavirus agents are still under research. Here, polyomavirus large tumor antigens (Tag) activities were identified as the viral target of cidofovir derivatives. The alkoxyalkyl-ester derivatives of cidofovir efficiently inhibit polyomavirus DNA replication in cell-free human extracts and a viral in vitro replication system only utilizing purified proteins. We present evidence that DNA helicase, and DNA binding activities of polyomavirus Tags are diminished in the presence of low concentrations of alkoxyalkyl-ester derivatives of cidofovir suggesting that the inhibition of viral DNA replication is at least in part mediated by inhibiting ssDNA and dsDNA binding activities of Tags. These findings show that the alkoxyalkyl-ester derivatives of cidofovir are effective in vitro without undergoing further conversions and conclude that the inhibitory mechanisms of nucleotide analog-based drugs are more complex than previously believed.

scholarly journals Amino-Terminal Domain Exchange Redirects Origin-Specific Interactions of Adeno-Associated Virus Rep78 In Vitro

2001 ◽  
Vol 75 (7) ◽  
pp. 3230-3239 ◽  
Author(s):  
Miran Yoon ◽  
Deborah H. Smith ◽  
Peter Ward ◽  
Francisco J. Medrano ◽  
Aneel K. Aggarwal ◽  
...  
Keyword(s):  
Dna Replication ◽  
Catalytic Domain ◽  
Adeno Associated Virus ◽  
Free System ◽  
Site Specific ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Site Specific Integration ◽  
Amino Terminal Domain

ABSTRACT The unique ability of adeno-associated virus type 2 (AAV) to site-specifically integrate its genome into a defined sequence on human chromosome 19 (AAVS1) makes it of particular interest for use in targeted gene delivery. The objective underlying this study is to provide evidence for the feasibility of retargeting site-specific integration into selected loci within the human genome. Current models postulate that AAV DNA integration is initiated through the interactions of the products of a single viral open reading frame,REP, with sequences present in AAVS1 that resemble the minimal origin for AAV DNA replication. Here, we present a cell-free system designed to dissect the Rep functions required to target site-specific integration using functional chimeric Rep proteins derived from AAV Rep78 and Rep1 of the closely related goose parvovirus. We show that amino-terminal domain exchange efficiently redirects the specificity of Rep to the minimal origin of DNA replication. Furthermore, we establish that the amino-terminal 208 amino acids of Rep78/68 constitute a catalytic domain of Rep sufficient to mediate site-specific endonuclease activity.

scholarly journals An emerging picture of FANCJ’s role in G4 resolution to facilitate DNA replication

NAR Cancer ◽  
2021 ◽  
Vol 3 (3) ◽  
Author(s):  
Robert M Brosh ◽  
Yuliang Wu
Keyword(s):  
Dna Replication ◽  
Double Helix ◽  
Dna Helicase ◽  
Nucleic Acid Binding ◽  
Dna Helicases ◽  
Recent Advances ◽  
G4 Dna ◽  
Cellular Dna

Abstract A well-accepted hallmark of cancer is genomic instability, which drives tumorigenesis. Therefore, understanding the molecular and cellular defects that destabilize chromosomal integrity is paramount to cancer diagnosis, treatment and cure. DNA repair and the replication stress response are overarching paradigms for maintenance of genomic stability, but the devil is in the details. ATP-dependent helicases serve to unwind DNA so it is replicated, transcribed, recombined and repaired efficiently through coordination with other nucleic acid binding and metabolizing proteins. Alternatively folded DNA structures deviating from the conventional anti-parallel double helix pose serious challenges to normal genomic transactions. Accumulating evidence suggests that G-quadruplex (G4) DNA is problematic for replication. Although there are multiple human DNA helicases that can resolve G4 in vitro, it is debated which helicases are truly important to resolve such structures in vivo. Recent advances have begun to elucidate the principal helicase actors, particularly in cellular DNA replication. FANCJ, a DNA helicase implicated in cancer and the chromosomal instability disorder Fanconi Anemia, takes center stage in G4 resolution to allow smooth DNA replication. We will discuss FANCJ’s role with its protein partner RPA to remove G4 obstacles during DNA synthesis, highlighting very recent advances and implications for cancer therapy.

scholarly journals Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation

2021 ◽  
Author(s):  
Allison W. McClure ◽  
John F.X. Diffley
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genotoxic Stress ◽  
Replication Initiation ◽  
Multiple Roles ◽  
Replication Forks ◽  
Dna Replication Stress ◽  
Necessary And Sufficient

SummaryThe Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

Sign in / Sign up

Export Citation Format

Share Document