The 3′-tail of a forked-duplex sterically determines whether one or two DNA strands pass through the central channel of a replication-fork helicase 1 1Edited by M. Gottesman

2000 ◽  
Vol 301 (2) ◽  
pp. 285-299 ◽  
Author(s):  
Daniel L Kaplan
Keyword(s):  
Replication Fork ◽  
Central Channel ◽  
Dna Strands ◽  
Pass Through

scholarly journals Dynamic Association of the Replication Initiator and Transcription Factor DnaA with the Bacillus subtilis Chromosome during Replication Stress

2008 ◽  
Vol 191 (2) ◽  
pp. 486-493 ◽  
Author(s):  
Adam M. Breier ◽  
Alan D. Grossman
Keyword(s):  
Transcription Factor ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Replication Fork ◽  
Replication Stress ◽  
Replication Initiation ◽  
Pass Through ◽  
Bacillus Subtilis Chromosome ◽  
Rapid Signaling ◽  
Replication Initiator

ABSTRACT DnaA functions as both a transcription factor and the replication initiator in bacteria. We characterized the DNA binding dynamics of DnaA on a genomic level. Based on cross-linking and chromatin immunoprecipitation data, DnaA binds at least 17 loci, 15 of which are regulated transcriptionally in response to inhibition of replication (replication stress). Six loci, each of which has a cluster of at least nine potential DnaA binding sites, had significant increases in binding by DnaA when replication was inhibited, indicating that the association of DnaA with at least some of its target sites is altered after replication stress. When replication resumed from oriC after inhibition of replication initiation, these high levels of binding decreased rapidly at origin-proximal and origin-distal regions, well before a replication fork could pass through each of the regulated regions. These findings indicate that there is rapid signaling to decrease activation of DnaA during replication and that interaction between DnaA bound at each site and the replication machinery is not required for regulation of DnaA activity in response to replication stress.

scholarly journals Single-molecule imaging of DNA gyrase activity in livingEscherichia coli

2018 ◽  
Author(s):  
Mathew Stracy ◽  
Adam J.M. Wollman ◽  
Elzbieta Kaja ◽  
Jacek Gapinski ◽  
Ji-Eun Lee ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Replication Fork ◽  
Dna Gyrase ◽  
Chromosomal Dna ◽  
Replication Fork Progression ◽  
Dna Strands ◽  
Steady State Levels ◽  
Negative Supercoiling ◽  
Supercoil Relaxation

ABSTRACTBacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in liveEscherichia colicells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with ∼12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of ∼2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was ∼8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.

scholarly journals Impaired Translesion Synthesis in Xeroderma Pigmentosum Variant Extracts

1999 ◽  
Vol 19 (3) ◽  
pp. 2206-2211 ◽  
Author(s):  
Agnes M. Cordonnier ◽  
Alan R. Lehmann ◽  
Robert P. P. Fuchs
Keyword(s):  
Xeroderma Pigmentosum ◽  
Replication Fork ◽  
Molecular Mechanisms ◽  
Translesion Synthesis ◽  
Distinct Pattern ◽  
Skin Fibroblasts ◽  
Primer Extension ◽  
Normal Cells ◽  
Replication Fork Progression ◽  
Dna Strands

ABSTRACT Xeroderma pigmentosum variant (XPV) cells are characterized by a cellular defect in the ability to synthesize intact daughter DNA strands on damaged templates. Molecular mechanisms that facilitate replication fork progression on damaged DNA in normal cells are not well defined. In this study, we used single-stranded plasmid molecules containing a single N-2-acetylaminofluorene (AAF) adduct to analyze translesion synthesis (TLS) catalyzed by extracts of either normal or XPV primary skin fibroblasts. In one of the substrates, the single AAF adduct was located at the 3′ end of a run of three guanines that was previously shown to induce deletion of one G by a slippage mechanism. Primer extension reactions performed by normal cellular extracts from four different individuals produced the same distinct pattern of TLS, with over 80% of the products resulting from the elongation of a slipped intermediate and the remaining 20% resulting from a nonslipped intermediate. In contrast, with cellular extracts from five different XPV patients, the TLS reaction was strongly reduced, yielding only low amounts of TLS via the nonslipped intermediate. With our second substrate, in which the AAF adduct was located at the first G in the run, thus preventing slippage from occurring, we confirmed that normal extracts were able to perform TLS 10-fold more efficiently than XPV extracts. These data demonstrate unequivocally that the defect in XPV cells resides in translesion synthesis independently of the slippage process.

Evidence from mutational specificity studies that yeast DNA polymerases δ and ϵ replicate different DNA strands at an intracellular replication fork 1 1Edited by A. R. Fersht

2000 ◽  
Vol 299 (2) ◽  
pp. 405-419 ◽  
Author(s):  
Ramachandran Karthikeyan ◽  
Edward J Vonarx ◽  
Andrew F.L Straffon ◽  
Michel Simon ◽  
Gérard Faye ◽  
...  
Keyword(s):  
Replication Fork ◽  
Dna Polymerases ◽  
Intracellular Replication ◽  
Mutational Specificity ◽  
Dna Strands

scholarly journals Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation

2017 ◽  
Vol 114 (5) ◽  
pp. E697-E706 ◽  
Author(s):  
Roxana Georgescu ◽  
Zuanning Yuan ◽  
Lin Bai ◽  
Ruda de Luna Almeida Santos ◽  
Jingchuan Sun ◽  
...  
Keyword(s):  
Replication Fork ◽  
Control Mechanism ◽  
The Other ◽  
Central Channel ◽  
Double Stranded Dna ◽  
Initial Melting ◽  
Quality Control Mechanism ◽  
Hexameric Helicases ◽  
Do So ◽  
Terminal Domains

The eukaryotic CMG (Cdc45, Mcm2–7, GINS) helicase consists of the Mcm2–7 hexameric ring along with five accessory factors. The Mcm2–7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EM single-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork. The duplex stem penetrates into the central channel of the N-tier and the unwound leading single-strand DNA traverses the channel through the N-tier into the C-tier motor, 5′-3′ through CMG. Therefore, the N-tier ring is pushed ahead by the C-tier ring during CMG translocation, opposite the currently accepted polarity. The polarity of the N-tier ahead of the C-tier places the leading Pol ε below CMG and Pol α-primase at the top of CMG at the replication fork. Surprisingly, the new N-tier to C-tier polarity of translocation reveals an unforeseen quality-control mechanism at the origin. Thus, upon assembly of head-to-head CMGs that encircle double-stranded DNA at the origin, the two CMGs must pass one another to leave the origin and both must remodel onto opposite strands of single-stranded DNA to do so. We propose that head-to-head motors may generate energy that underlies initial melting at the origin.

scholarly journals Structures and operating principles of the replisome

Science ◽  
2019 ◽  
Vol 363 (6429) ◽  
pp. eaav7003 ◽  
Author(s):  
Yang Gao ◽  
Yanxiang Cui ◽  
Tara Fox ◽  
Shiqiang Lin ◽  
Huaibin Wang ◽  
...  
Keyword(s):  
Replication Fork ◽  
Model System ◽  
Molecular Organization ◽  
Cryo Electron Microscopy ◽  
Adenosine Triphosphatases ◽  
Dna Strands ◽  
Leading Strand ◽  
Dna Strand ◽  
Aaa Atpases ◽  
Lagging Strand

Visualization in atomic detail of the replisome that performs concerted leading– and lagging–DNA strand synthesis at a replication fork has not been reported. Using bacteriophage T7 as a model system, we determined cryo–electron microscopy structures up to 3.2-angstroms resolution of helicase translocating along DNA and of helicase-polymerase-primase complexes engaging in synthesis of both DNA strands. Each domain of the spiral-shaped hexameric helicase translocates sequentially hand-over-hand along a single-stranded DNA coil, akin to the way AAA+ ATPases (adenosine triphosphatases) unfold peptides. Two lagging-strand polymerases are attached to the primase, ready for Okazaki fragment synthesis in tandem. A β hairpin from the leading-strand polymerase separates two parental DNA strands into a T-shaped fork, thus enabling the closely coupled helicase to advance perpendicular to the downstream DNA duplex. These structures reveal the molecular organization and operating principles of a replisome.

scholarly journals DNA Polymerases at the Eukaryotic Replication Fork Thirty Years after: Connection to Cancer

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3489
Author(s):  
Youri I. Pavlov ◽  
Anna S. Zhuk ◽  
Elena I. Stepchenkova
Keyword(s):  
Replication Fork ◽  
Genome Instability ◽  
Dna Polymerases ◽  
Structure And Function ◽  
The Past ◽  
New Findings ◽  
Dna Strands ◽  
Leading Strand ◽  
And Function

Recent studies on tumor genomes revealed that mutations in genes of replicative DNA polymerases cause a predisposition for cancer by increasing genome instability. The past 10 years have uncovered exciting details about the structure and function of replicative DNA polymerases and the replication fork organization. The principal idea of participation of different polymerases in specific transactions at the fork proposed by Morrison and coauthors 30 years ago and later named “division of labor,” remains standing, with an amendment of the broader role of polymerase δ in the replication of both the lagging and leading DNA strands. However, cancer-associated mutations predominantly affect the catalytic subunit of polymerase ε that participates in leading strand DNA synthesis. We analyze how new findings in the DNA replication field help elucidate the polymerase variants’ effects on cancer.

scholarly journals THE NUCLEAR ANNULI AS PATHWAYS FOR NUCLEOCYTOPLASMIC EXCHANGES

1962 ◽  
Vol 14 (1) ◽  
pp. 65-72 ◽  
Author(s):  
Carl M. Feldherr
Keyword(s):  
Electron Microscopy ◽  
Nuclear Envelope ◽  
Colloidal Gold ◽  
Colloidal Particles ◽  
Time Interval ◽  
Central Channel ◽  
Time Intervals ◽  
Gold Particles ◽  
Pass Through ◽  
Individual Particles

Colloidal gold particles, 25 to 55 A in diameter, which had been coated with polyvinylpyrrolidone, were microinjected into the ground cytoplasm of amebas (Chaos chaos). At time intervals of 1 minute, 2 minutes, 10 minutes, and 24 hours after injection the cells were fixed for electron microscopy. After 24 hours, gold particles were found in both the nuclei and the ground cytoplasm, the concentration being higher in the nuclei. Colloidal particles were also present in the nuclei after 10 minutes, but at this time interval the concentration did not appear to be greater than that in the ground cytoplasm. One and 2 minutes after injection, the gold particles were located almost exclusively in the ground cytoplasm; however, individual particles were often found within the annuli of the nuclear envelope, and were located specifically in the centers of these structures. The results suggest that at least some of the gold particles which enter the nuclei pass through the annuli, and that passage through these structures may be restricted to a central channel.

Functional interaction between Fanconi anemia and mTOR pathways during stalled replication fork recovery

2020 ◽  
Author(s):  
Matthew Nolan ◽  
Kenneth Knudson ◽  
Marina K Holz ◽  
Indrajit Chaudhury
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Fanconi Anemia ◽  
Replication Fork ◽  
Genomic Stability ◽  
Repair Pathway ◽  
Functional Interaction ◽  
Mechanistic Target Of Rapamycin ◽  
Dna Strands ◽  
Repair Pathways

We have previously demonstrated that Fanconi Anemia (FA) proteins work in concert with other FA and non-FA proteins to mediate stalled replication fork restart. Previous studies suggest a connection between FA protein FANCD2 and a non-FA protein mechanistic target of rapamycin (mTOR). A recent study showed that mTOR is involved in actin-dependent DNA replication fork restart, suggesting possible roles in FA DNA repair pathway. In this study, we demonstrate that during replication stress mTOR interacts and cooperates with FANCD2 to provide cellular stability, mediates stalled replication fork restart and prevents nucleolytic degradation of the nascent DNA strands. Taken together, this study unravels a novel functional cross-talk between two important mechanisms: mTOR and FA DNA repair pathways that ensure genomic stability.

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