Supporting evidence for DNA shearing as a mechanism for origin unwinding in eukaryotes

Langston, Lance D.; Georgescu, Roxana E.; O’Donnell, Michael

doi:10.1101/739557

Cited by 3 publications

(3 citation statements)

References 33 publications

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“…4 ). This result strongly supports the idea that T-Ag interacts with the negatively charged phosphate backbone of the tracking strand during duplex DNA translocation, similar to what was observed for CMG tracking on dsDNA ( 40 , 57 ). Somewhat surprisingly, the presence of MePs on the nontracking strand increased unwinding twofold compared with the substrate with no MePs ( Fig.…”

Section: Resultssupporting

confidence: 88%

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SV40 T-antigen uses a DNA shearing mechanism to initiate origin unwinding

Langston

Yuan

Georgescu

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Duplication of DNA genomes requires unwinding of the double-strand (ds) DNA so that each single strand (ss) can be copied by a DNA polymerase. The genomes of eukaryotic cells are unwound by two ring-shaped hexameric helicases that initially encircle dsDNA but transition to ssDNA for function as replicative helicases. How the duplex is initially unwound, and the role of the two helicases in this process, is poorly understood. We recently described an initiation mechanism for eukaryotes in which the two helicases are directed inward toward one another and shear the duplex open by pulling on opposite strands of the duplex while encircling dsDNA [L. D. Langston, M. E. O'Donnell, eLife 8 , e46515 (2019)]. Two head-to-head T-Antigen helicases are long known to be loaded at the SV40 origin. We show here that T-Antigen tracks head (N-tier) first on ssDNA, opposite the direction proposed for decades. We also find that SV40 T-Antigen tracks directionally while encircling dsDNA and mainly tracks on one strand of the duplex in the same orientation as during ssDNA translocation. Further, two inward directed T-Antigen helicases on dsDNA are able to melt a 150-bp duplex. These findings explain the “rabbit ear” DNA loops observed at the SV40 origin by electron microscopy and reconfigure how the DNA loops emerge from the double hexamer relative to earlier models. Thus, the mechanism of DNA shearing by two opposing helicases is conserved in a eukaryotic viral helicase and may be widely used to initiate origin unwinding of dsDNA genomes.

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Section: Resultssupporting

confidence: 88%

“…We have also demonstrated that the head-to-head orientation of the two CMGs enables them to rip, or shear, the duplex apart, similar to the model of Fig. 6 B and proposed here for T-Ag ( 40 , 57 ).…”

Section: Discussionsupporting

confidence: 81%

SV40 T-antigen uses a DNA shearing mechanism to initiate origin unwinding

Langston

Yuan

Georgescu

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…This conundrum has recently been solved. Briefly, the two CMGs push inward toward one another by pulling on opposite strands of the duplex, and this generates sufficient torque to separate the duplex into single-strands [ 62 , 63 ]. The CMGs then use a ssDNA gate within the Mcm2–7 ring to expel the lagging strand, as demonstrated in recent single-molecule studies [ 64 ], and in biochemical ensemble studies [ 23 ].…”

Section: Cmg Tracks On Dna N-tier First At a Replication Forkmentioning

confidence: 99%

Anatomy of a twin DNA replication factory

Yao

O'Donnell

2020

Biochemical Society Transactions

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The replication of DNA in chromosomes is initiated at sequences called origins at which two replisome machines are assembled at replication forks that move in opposite directions. Interestingly, in vivo studies observe that the two replication forks remain fastened together, often referred to as a replication factory. Replication factories containing two replisomes are well documented in cellular studies of bacteria (Escherichia coli and Bacillus subtilis) and the eukaryote, Saccharomyces cerevisiae. This basic twin replisome factory architecture may also be preserved in higher eukaryotes. Despite many years of documenting the existence of replication factories, the molecular details of how the two replisome machines are tethered together has been completely unknown in any organism. Recent structural studies shed new light on the architecture of a eukaryote replisome factory, which brings with it a new twist on how a replication factory may function.

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Mechanism of eukaryotic origin unwinding is a dual helicase DNA shearing process

Langston,

Georgescu,

O'Donnell

2023

Proc. Natl. Acad. Sci. U.S.A.

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DNA replication in all cells begins with the melting of base pairs at the duplex origin to allow access to single-stranded DNA templates which are replicated by DNA polymerases. In bacteria, origin DNA is presumed to be melted by accessory proteins that allow loading of two ring-shaped replicative helicases around single-strand DNA (ssDNA) for bidirectional unwinding and DNA replication. In eukaryotes, by contrast, two replicative CMG (Cdc45-Mcm2-7-GINS) helicases are initially loaded head to head around origin double-strand DNA (dsDNA), and there does not appear to be a separate origin unwinding factor. This led us to investigate whether head-to-head CMGs use their adenosine triphosphate (ATP)-driven motors to initiate duplex DNA unwinding at the origin. Here, we show that CMG tracks on one strand of the duplex while surrounding it, and this feature allows two head-to-head CMGs to unwind dsDNA by using their respective motors to pull on opposite strands of the duplex. We further show that while CMG is capable of limited duplex unwinding on its own, the extent of unwinding is greatly and rapidly stimulated by addition of the multifunctional CMG-binding protein Mcm10 that is critical for productive initiation of DNA replication in vivo. On the basis of these findings, we propose that Mcm10 is a processivity or positioning factor that helps translate the work performed by the dual CMG motors at the origin into productive unwinding that facilitates bidirectional DNA replication.

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Supporting evidence for DNA shearing as a mechanism for origin unwinding in eukaryotes

Cited by 3 publications

References 33 publications

SV40 T-antigen uses a DNA shearing mechanism to initiate origin unwinding

SV40 T-antigen uses a DNA shearing mechanism to initiate origin unwinding

Anatomy of a twin DNA replication factory

Mechanism of eukaryotic origin unwinding is a dual helicase DNA shearing process

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