Replicative Helicases as the Central Organizing Motor Proteins in the Molecular Machines of the Elongating Eukaryotic Replication Fork

Nuclease dead Cas9 is a programmable roadblock for DNA replication

10.1101/455543 ◽

2018 ◽

Cited By ~ 1

Author(s):

Kelsey Whinn ◽

Gurleen Kaur ◽

Jacob S. Lewis ◽

Grant Schauer ◽

Stefan Müller ◽

...

Keyword(s):

Dna Replication ◽

Single Molecule ◽

Replication Fork ◽

Molecular Machines ◽

Replication Forks ◽

Chromosomal Dna ◽

Single Molecule Level ◽

Replication Fork Arrest

DNA replication occurs on chromosomal DNA while processes such as DNA repair, recombination and transcription continue. However, we have limited experimental tools to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct fused to the photo-stable dL5 protein fluoromodule as a novel, targetable protein-DNA roadblock for studying replication fork arrest at the single-molecule level in vitro as well as in vivo. We find that the specifically bound dCas9–guideRNA complex arrests viral, bacterial and eukaryotic replication forks in vitro.

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Nuclease dead Cas9 is a programmable roadblock for DNA replication

Scientific Reports ◽

10.1038/s41598-019-49837-z ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 10

Author(s):

Kelsey S. Whinn ◽

Gurleen Kaur ◽

Jacob S. Lewis ◽

Grant D. Schauer ◽

Stefan H. Mueller ◽

...

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Molecular Machines ◽

Enzymatic Activities ◽

Replication Forks ◽

Dna Substrates ◽

Replication Fork Arrest

Abstract Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9–guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro.

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Mechanism of replication fork reversal and protection by human RAD51 and RAD51 paralogs

10.21203/rs.3.rs-1096289/v1 ◽

2021 ◽

Author(s):

Petr Cejka ◽

Swagata Halder ◽

Aurore Sanchez ◽

Lepakshi Ranjha ◽

Angelo Taglialatela ◽

...

Keyword(s):

Initial Function ◽

Replication Fork ◽

Motor Proteins ◽

Replication Forks ◽

Branch Migration ◽

Dna Protection ◽

Protective Function ◽

Double Stranded Dna ◽

Low Concentrations ◽

Redundant Functions

Abstract SMARCAL1, ZRANB3 and HLTF are all required for the remodeling of replication forks upon stress. Using reconstituted reactions, we show that the motor proteins have unequal biochemical capacities, explaining why they have non-redundant functions. Whereas SMARCAL1 uniquely anneals RPA-coated ssDNA, suggesting an initial function in fork reversal, it becomes comparatively inefficient in subsequent branch migration. We also show that low concentrations of RAD51 and the RAD51 paralog complex, RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2), directly stimulate SMARCAL1 and ZRANB3 but not HLTF, providing a mechanism underlying previous cellular data implicating these factors in fork reversal. Upon reversal, RAD51 protects replication forks from degradation by MRE11, DNA2 and EXO1 nucleases. We show that the protective function of RAD51 unexpectedly depends on its binding to double-stranded DNA, and higher RAD51 concentrations are required for DNA protection compared to reversal. Together, we define the non-canonical functions of RAD51 and its paralogs in replication fork reversal and protection.

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Unwinding of a DNA replication fork by a hexameric viral helicase

Nature Communications ◽

10.1038/s41467-021-25843-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Abid Javed ◽

Balazs Major ◽

Jonathan A. Stead ◽

Cyril M. Sanders ◽

Elena V. Orlova

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Motor Proteins ◽

Full Length ◽

Base Pairs ◽

Binding Domains ◽

Double Stranded Dna ◽

Viral Helicase ◽

Hexameric Helicases ◽

Auxiliary Role

AbstractHexameric helicases are motor proteins that unwind double-stranded DNA (dsDNA) during DNA replication but how they are optimised for strand separation is unclear. Here we present the cryo-EM structure of the full-length E1 helicase from papillomavirus, revealing all arms of a bound DNA replication fork and their interactions with the helicase. The replication fork junction is located at the entrance to the helicase collar ring, that sits above the AAA + motor assembly. dsDNA is escorted to and the 5´ single-stranded DNA (ssDNA) away from the unwinding point by the E1 dsDNA origin binding domains. The 3´ ssDNA interacts with six spirally-arranged β-hairpins and their cyclical top-to-bottom movement pulls the ssDNA through the helicase. Pulling of the RF against the collar ring separates the base-pairs, while modelling of the conformational cycle suggest an accompanying movement of the collar ring has an auxiliary role, helping to make efficient use of ATP in duplex unwinding.

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Replication Fork Stalling at Natural Impediments

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00030-06 ◽

2007 ◽

Vol 71 (1) ◽

pp. 13-35 ◽

Cited By ~ 330

Author(s):

Ekaterina V. Mirkin ◽

Sergei M. Mirkin

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Genetic Factors ◽

Genomic Stability ◽

Molecular Machines ◽

Dna Binding Proteins ◽

Replication Forks ◽

Fork Stalling ◽

Transcription Complexes

SUMMARY Accurate and complete replication of the genome in every cell division is a prerequisite of genomic stability. Thus, both prokaryotic and eukaryotic replication forks are extremely precise and robust molecular machines that have evolved to be up to the task. However, it has recently become clear that the replication fork is more of a hurdler than a runner: it must overcome various obstacles present on its way. Such obstacles can be called natural impediments to DNA replication, as opposed to external and genetic factors. Natural impediments to DNA replication are particular DNA binding proteins, unusual secondary structures in DNA, and transcription complexes that occasionally (in eukaryotes) or constantly (in prokaryotes) operate on replicating templates. This review describes the mechanisms and consequences of replication stalling at various natural impediments, with an emphasis on the role of replication stalling in genomic instability.

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