The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader

ABSTRACTIn many bacteria and in eukaryotes, replication fork establishment requires the controlled loading of hexameric, ring-shaped helicases around DNA by AAA+ ATPases. How loading factors use ATP to control helicase deposition is poorly understood. Here, we dissect how specific ATPase elements of E. coli DnaC, an archetypal loader for the bacterial DnaB helicase, play distinct roles in helicase loading and the activation of DNA unwinding. We identify a new element, the arginine-coupler, which regulates the switch-like behavior of DnaC to prevent futile ATPase cycling and maintains loader responsiveness to replication restart systems. Our data help explain how the ATPase cycle of a AAA+-family helicase loader is channeled into productive action on its target; comparative studies indicate elements analogous to the Arg-coupler are present in related, switch-like AAA+ proteins that control replicative helicase loading in eukaryotes, as well as polymerase clamp loading and certain classes of DNA transposases.

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The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader

eLife ◽

10.7554/elife.64232 ◽

2021 ◽

Vol 10 ◽

Author(s):

Neha Puri ◽

Amy J Fernandez ◽

Valerie L O'Shea Murray ◽

Sarah McMillan ◽

James L Keck ◽

...

Keyword(s):

Replication Fork ◽

Substrate Recognition ◽

Dna Unwinding ◽

Replicative Helicase ◽

E Coli ◽

Helicase Loading ◽

Dnab Helicase ◽

Replication Restart ◽

Clamp Loading ◽

Aaa Atpases

In many bacteria and in eukaryotes, replication fork establishment requires the controlled loading of hexameric, ring-shaped helicases around DNA by AAA+ ATPases. How loading factors use ATP to control helicase deposition is poorly understood. Here, we dissect how specific ATPase elements of E. coli DnaC, an archetypal loader for the bacterial DnaB helicase, play distinct roles in helicase loading and the activation of DNA unwinding. We identify a new element, the arginine-coupler, which regulates the switch-like behavior of DnaC to prevent futile ATPase cycling and maintains loader responsiveness to replication restart systems. Our data help explain how the ATPase cycle of a AAA+-family helicase loader is channeled into productive action on its target; comparative studies indicate elements analogous to the Arg-coupler are present in related, switch-like AAA+ proteins that control replicative helicase loading in eukaryotes, as well as polymerase clamp loading and certain classes of DNA transposases.

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Replication Fork Breakage and Restart inEscherichia coli

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00013-18 ◽

2018 ◽

Vol 82 (3) ◽

Cited By ~ 24

Author(s):

Bénédicte Michel ◽

Anurag K. Sinha ◽

David R. F. Leach

Keyword(s):

Replication Fork ◽

Strand Break ◽

Replication Forks ◽

Wild Type ◽

Dsb Repair ◽

Replicative Helicase ◽

Content Type ◽

E Coli ◽

Double Stranded Dna ◽

Replication Restart

SUMMARYIn all organisms, replication impairments are an important source of genome rearrangements, mainly because of the formation of double-stranded DNA (dsDNA) ends at inactivated replication forks. Three reactions for the formation of dsDNA ends at replication forks were originally described forEscherichia coliand became seminal models for all organisms: the encounter of replication forks with preexisting single-stranded DNA (ssDNA) interruptions, replication fork reversal, and head-to-tail collisions of successive replication rounds. Here, we first review the experimental evidence that now allows us to know when, where, and how these three different reactions occur inE. coli. Next, we recall our recent studies showing that in wild-typeE. coli, spontaneous replication fork breakage occurs in 18% of cells at each generation. We propose that it results from the replication of preexisting nicks or gaps, since it does not involve replication fork reversal or head-to-tail fork collisions. In therecBmutant, deficient for double-strand break (DSB) repair, fork breakage triggers DSBs in the chromosome terminus during cell division, a reaction that is heritable for several generations. Finally, we recapitulate several observations suggesting that restart from intact inactivated replication forks and restart from recombination intermediates require different sets of enzymatic activities. The finding that 18% of cells suffer replication fork breakage suggests that DNA remains intact at most inactivated forks. Similarly, only 18% of cells need the helicase loader for replication restart, which leads us to speculate that the replicative helicase remains on DNA at intact inactivated replication forks and is reactivated by the replication restart proteins.

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Mechanism of RPA-Facilitated Processive DNA Unwinding by the Eukaryotic CMG Helicase

10.1101/796003 ◽

2019 ◽

Author(s):

Hazal B. Kose ◽

Sherry Xie ◽

George Cameron ◽

Melania S. Strycharska ◽

Hasan Yardimci

Keyword(s):

Single Molecule ◽

Replication Fork ◽

Double Helix ◽

Dna Unwinding ◽

Helicase Activity ◽

Replicative Helicase ◽

Double Stranded Dna ◽

Order Of Magnitude ◽

Dna Strand ◽

Dna Double Helix

AbstractThe DNA double helix is unwound by the Cdc45/Mcm2-7/GINS (CMG) complex at the eukaryotic replication fork. While isolated CMG unwinds duplex DNA very slowly, its fork unwinding rate is stimulated by an order of magnitude by single-stranded DNA binding protein, RPA. However, the molecular mechanism by which RPA enhances CMG helicase activity remained elusive. Here, we demonstrate that engagement of CMG with parental double-stranded DNA (dsDNA) at the replication fork impairs its helicase activity, explaining the slow DNA unwinding by isolated CMG. Using single-molecule and ensemble biochemistry, we show that binding of RPA to the excluded DNA strand prevents duplex engagement by the helicase and speeds up CMG-mediated DNA unwinding. When stalled due to dsDNA interaction, DNA rezipping-induced helicase backtracking re-establishes productive helicase-fork engagement underscoring the significance of plasticity in helicase action. Together, our results elucidate the dynamics of CMG at the replication fork and reveal how other replisome components can mediate proper DNA engagement by the replicative helicase to achieve efficient fork progression.

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Viral hijacking of a replicative helicase loader and its implications for helicase loading control and phage replication

eLife ◽

10.7554/elife.14158 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 6

Author(s):

Iris V Hood ◽

James M Berger

Keyword(s):

Self Assembly ◽

New Approach ◽

Replicative Helicase ◽

Aaa Atpase ◽

Helicase Loading ◽

Phage Protein ◽

Biochemical Analyses ◽

Viral Hijacking ◽

Aaa Atpases ◽

Hexameric Helicases

Replisome assembly requires the loading of replicative hexameric helicases onto origins by AAA+ ATPases. How loader activity is appropriately controlled remains unclear. Here, we use structural and biochemical analyses to establish how an antimicrobial phage protein interferes with the function of the Staphylococcus aureus replicative helicase loader, DnaI. The viral protein binds to the loader’s AAA+ ATPase domain, allowing binding of the host replicative helicase but impeding loader self-assembly and ATPase activity. Close inspection of the complex highlights an unexpected locus for the binding of an interdomain linker element in DnaI/DnaC-family proteins. We find that the inhibitor protein is genetically coupled to a phage-encoded homolog of the bacterial helicase loader, which we show binds to the host helicase but not to the inhibitor itself. These findings establish a new approach by which viruses can hijack host replication processes and explain how loader activity is internally regulated to prevent aberrant auto-association.

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Identification of a domain of Escherichia coli primase required for functional interaction with the DnaB helicase at the replication fork.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)41829-1 ◽

1994 ◽

Vol 269 (6) ◽

pp. 4675-4682 ◽

Cited By ~ 2

Author(s):

K. Tougu ◽

H. Peng ◽

K.J. Marians

Keyword(s):

Escherichia Coli ◽

Replication Fork ◽

Functional Interaction ◽

Dnab Helicase

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

Journal of Biological Chemistry ◽

10.1074/jbc.m407772200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45586-45593 ◽

Cited By ~ 30

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.

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E. coli DnaB Helicase Stimulates E. coli Primase Activity and Alters Its Initiation Specificity

Biochemical Society Transactions ◽

10.1042/bst028a248b ◽

2000 ◽

Vol 28 (5) ◽

pp. A248-A248

Author(s):

M. A. Griep ◽

S. Bhattacharyya ◽

S. K. Johnson

Keyword(s):

E Coli ◽

Dnab Helicase

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Replication fork stalling elicits chromatin compaction for the stability of stalling replication forks

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1821475116 ◽

2019 ◽

Vol 116 (29) ◽

pp. 14563-14572 ◽

Cited By ~ 3

Author(s):

Gang Feng ◽

Yue Yuan ◽

Zeyang Li ◽

Lu Wang ◽

Bo Zhang ◽

...

Keyword(s):

Replication Fork ◽

Cellular Mechanism ◽

Eukaryotic Cells ◽

Replication Forks ◽

Physical Separation ◽

Replicative Helicase ◽

Chromatin Compaction ◽

Acetylation Site ◽

Fork Stalling ◽

The Stability

DNA replication forks in eukaryotic cells stall at a variety of replication barriers. Stalling forks require strict cellular regulations to prevent fork collapse. However, the mechanism underlying these cellular regulations is poorly understood. In this study, a cellular mechanism was uncovered that regulates chromatin structures to stabilize stalling forks. When replication forks stall, H2BK33, a newly identified acetylation site, is deacetylated and H3K9 trimethylated in the nucleosomes surrounding stalling forks, which results in chromatin compaction around forks. Acetylation-mimic H2BK33Q and its deacetylase clr6-1 mutations compromise this fork stalling-induced chromatin compaction, cause physical separation of replicative helicase and DNA polymerases, and significantly increase the frequency of stalling fork collapse. Furthermore, this fork stalling-induced H2BK33 deacetylation is independent of checkpoint. In summary, these results suggest that eukaryotic cells have developed a cellular mechanism that stabilizes stalling forks by targeting nucleosomes and inducing chromatin compaction around stalling forks. This mechanism is named the “Chromsfork” control: Chromatin Compaction Stabilizes Stalling Replication Forks.

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Unwinding 20 Years of the Archaeal Minichromosome Maintenance Helicase

Journal of Bacteriology ◽

10.1128/jb.00729-19 ◽

2020 ◽

Vol 202 (6) ◽

Cited By ~ 1

Author(s):

Lori M. Kelman ◽

William B. O’Dell ◽

Zvi Kelman

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Duplex Dna ◽

Chromosomal Dna ◽

Minichromosome Maintenance ◽

Replicative Helicase ◽

Dna Helicases ◽

First Report ◽

20Th Anniversary

ABSTRACT Replicative DNA helicases are essential cellular enzymes that unwind duplex DNA in front of the replication fork during chromosomal DNA replication. Replicative helicases were discovered, beginning in the 1970s, in bacteria, bacteriophages, viruses, and eukarya, and, in the mid-1990s, in archaea. This year marks the 20th anniversary of the first report on the archaeal replicative helicase, the minichromosome maintenance (MCM) protein. This minireview summarizes 2 decades of work on the archaeal MCM.

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