scholarly journals Rad53 controls DNA unwinding after helicase-polymerase uncoupling at DNA replication forks

2019 ◽  
Author(s):  
Sujan Devbhandari ◽  
Dirk Remus
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Point Mutations ◽  
Dna Helicase ◽  
Dna Unwinding ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Replication Checkpoint ◽  
Polymerase Domain ◽  
Tightly Coupled

ABSTRACTThe coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Using the reconstituted budding yeast DNA replication system, we demonstrate that Pol ε variants harboring catalytic point mutations in the Pol2 polymerase domain, contrary to Pol2 polymerase domain deletions, inhibit DNA synthesis at replication forks by displacing Pol δ from PCNA/primer-template junctions, causing excessive DNA unwinding by the replicative DNA helicase, CMG, uncoupled from DNA synthesis. Mutations that suppress the inhibition of Pol δ by Pol ε restore viability in Pol2 polymerase point mutant cells. We also observe uninterrupted DNA unwinding at replication forks upon dNTP depletion or chemical inhibition of DNA polymerases, demonstrating that leading strand synthesis is not tightly coupled to DNA unwinding by CMG. Importantly, the Rad53 kinase controls excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork-stabilization by the replication checkpoint.

scholarly journals Synthetic DNA Replication Bubbles Bound and Unwound with Twofold Symmetry by a Simian Virus 40 T-Antigen Double Hexamer

1998 ◽  
Vol 72 (11) ◽  
pp. 8676-8681 ◽  
Author(s):  
Natalia V. Smelkova ◽  
James A. Borowiec
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Helicase ◽  
T Antigen ◽  
Dna Unwinding ◽  
Replication Forks ◽  
Helicase Activity ◽  
Synthetic Dna ◽  
Stimulation Of

ABSTRACT Dimerization of simian virus 40 T-antigen hexamers (TAgH) into double hexamers (TAgDH) on model DNA replication forks has been found to greatly stimulate T-antigen DNA helicase activity. To explore the interaction of TAgDH with DNA during unwinding, we examined the binding of TAgDH to synthetic DNA replication bubbles. Tests of replication bubble substrates containing different single-stranded DNA (ssDNA) lengths indicated that efficient formation of a TAgDH requires ≥40 nucleotides (nt) of ssDNA. DNase I probing of a substrate containing a 60-nt ssDNA bubble complexed with a TAgDH revealed that T antigen bound the substrate with twofold symmetry. The strongest protection was observed over the 5′ junction on each strand, with 5 bp of duplex DNA and ∼17 nt of adjacent ssDNA protected from nuclease cleavage. Stimulation of the T-antigen DNA helicase activity by an increase in ATP concentration caused the protection to extend in the 5′ direction into the duplex region, while resulting in no significant changes to the 3′ edge of strongest protection. Our data indicate that each TAgH encircles one ssDNA strand, with a different strand bound at each junction. The process of DNA unwinding results in each TAgH interacting with a greater length of DNA than was initially bound, suggesting the generation of a more highly processive helicase complex.

scholarly journals Examination of the roles of a conserved motif in the PriA helicase in structure-specific DNA unwinding and processivity

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0255409
Author(s):  
Alexander T. Duckworth ◽  
Tricia A. Windgassen ◽  
James L. Keck
Keyword(s):  
Dna Replication ◽  
Dna Helicase ◽  
Sequence Motif ◽  
Dna Unwinding ◽  
Helicase Domain ◽  
Replication Forks ◽  
Dna Helicases ◽  
Conserved Sequence ◽  
Replication Restart ◽  
Dna Replication Restart

DNA replication complexes (replisomes) frequently encounter barriers that can eject them prematurely from the genome. To avoid the lethality of incomplete DNA replication that arises from these events, bacteria have evolved “DNA replication restart” mechanisms to reload replisomes onto abandoned replication forks. The Escherichia coli PriA DNA helicase orchestrates this process by recognizing and remodeling replication forks and recruiting additional proteins that help to drive replisome reloading. We have identified a conserved sequence motif within a linker region of PriA that docks into a groove on the exterior of the PriA helicase domain. Alterations to the motif reduce the apparent processivity and attenuate structure-specific helicase activity in PriA, implicating the motif as a potential autoregulatory element in replication fork processing. The study also suggests that multiple PriA molecules may function in tandem to enhance DNA unwinding processivity, highlighting an unexpected similarity between PriA and other DNA helicases.

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.

Single-molecule binding characterization of primosomal protein PriA involved in replication restart

2021 ◽  
Author(s):  
Tzu-Yu Lee ◽  
Yi-Ching Li ◽  
Min-Guan Lin ◽  
Chwan-Deng Hsiao ◽  
Hung-Wen Li
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Single Molecule ◽  
Replication Forks ◽  
Bacterial Survival ◽  
Dna Damages ◽  
Replication Restart

DNA damages lead to stalled or collapsed replication forks. Replication restart primosomes re-initiate DNA synthesis at these stalled or collapsed DNA replication forks, which is important for bacterial survival. Primosomal...

Stimulation of DNA Synthesis by Mouse DNA Helicase B in a DNA Replication System Containing Eukaryotic Replication Origins

Biochemistry ◽  
1995 ◽  
Vol 34 (24) ◽  
pp. 7913-7922 ◽  
Author(s):  
Ken Matsumoto ◽  
Masayuki Seki ◽  
Chikahide Masutani ◽  
Shusuke Tada ◽  
Takemi Enomoto ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Helicase ◽  
Replication Origins ◽  
Replication System ◽  
Stimulation Of

Method of mapping DNA replication origins

1985 ◽  
Vol 5 (1) ◽  
pp. 85-92
Author(s):  
L D Spotila ◽  
J A Huberman
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Replication Forks ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Dna Restriction Fragments ◽  
Dna Restriction ◽  
Dna Replication Origins

We have developed a method which allows determination of the direction in which replication forks move through segments of chromosomal DNA for which cloned probes are available. The method is based on the facts that DNA restriction fragments containing replication forks migrate more slowly through agarose gels than do non-fork-containing fragments and that the extent of retardation of the fork-containing fragments is a function of the extent of replication. The procedure allows the identification of DNA replication origins as sites from which replication forks diverge. In this paper we demonstrate the feasibility of this procedure, with simian virus 40 DNA as a model, and we discuss its applicability to other systems.

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

2009 ◽  
Vol 187 (1) ◽  
pp. 15-23 ◽  
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.

scholarly journals A Dominant-negative Mutant of Human DNA Helicase B Blocks the Onset of Chromosomal DNA Replication

2002 ◽  
Vol 277 (43) ◽  
pp. 40853-40861 ◽  
Author(s):  
Poonam Taneja ◽  
Jinming Gu ◽  
Rui Peng ◽  
Ryan Carrick ◽  
Fumiaki Uchiumi ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Helicase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Chromosomal Dna ◽  
Human Dna ◽  
Negative Mutant

scholarly journals Functional Requirement of Noncoding Y RNAs for Human Chromosomal DNA Replication

2006 ◽  
Vol 26 (18) ◽  
pp. 6993-7004 ◽  
Author(s):  
Christo P. Christov ◽  
Timothy J. Gardiner ◽  
Dávid Szüts ◽  
Torsten Krude
Keyword(s):  
Dna Replication ◽  
Functional Characterization ◽  
Noncoding Rnas ◽  
Biological Processes ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Free System ◽  
Specific Degradation

ABSTRACT Noncoding RNAs are recognized increasingly as important regulators of fundamental biological processes, such as gene expression and development, in eukaryotes. We report here the identification and functional characterization of the small noncoding human Y RNAs (hY RNAs) as novel factors for chromosomal DNA replication in a human cell-free system. In addition to protein fractions, hY RNAs are essential for the establishment of active chromosomal DNA replication forks in template nuclei isolated from late-G1-phase human cells. Specific degradation of hY RNAs leads to the inhibition of semiconservative DNA replication in late-G1-phase template nuclei. This inhibition is negated by resupplementation of hY RNAs. All four hY RNAs (hY1, hY3, hY4, and hY5) can functionally substitute for each other in this system. Mutagenesis of hY1 RNA showed that the binding site for Ro60 protein, which is required for Ro RNP assembly, is not essential for DNA replication. Degradation of hY1 RNA in asynchronously proliferating HeLa cells by RNA interference reduced the percentages of cells incorporating bromodeoxyuridine in vivo. These experiments implicate a functional role for hY RNAs in human chromosomal DNA replication.

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