Monday, February 9, 2009

Polymerase Dynamics at the Eukaryotic DNA Replication Fork



Renowned French scientist and 1965 Nobel laureate Jacques Monod once said "What's true for E. coli is true for an elephant".
The mechanistic basis for DNA replication is partially an example of this. The extensive studies made on E. coli revealed how DNA is copied, the series of proteins involved, and how it solves the apparent 'problem' of the DNA being in an antiparallel disposition, considering that the polymerase has a polarity (the 'trombone' model. See attached video.). The mechanism (which entails recognition of the origin of replication, assembly of the replication complex, loading of the clamp, etc.) was later shown to be essentially the same in eukaryotes, although almost always involving larger complexes and more proteins (See O’ Donnell’s review for details 1).
Despite the mechanistic similarities between the DNA replication process between prokaryotes and eukaryotes, a particular difference (among many others) involves the polymerases participating in primer synthesis and elongation of the leading and lagging strand.
In E. coli, the primer is synthesized by the product of dnaG, primase, and both the leading and lagging strand are synthesized by polIII core, in the context of a holoenzyme composed of a series of subunits.
In eukaryotes, the primer on the origins (which are multiple in these organisms, in contrast to E. coli, where there is a single origin of replication) is synthesized by DNA Pol α/primase and the leading and lagging strands have been suggested to be synthesized by different polymerases; DNA polymerase{delta} would synthesize the lagging strand and DNA polymerase {epsilon} the leading.

This review discusses evidence that support this model of different polymerases acting on the leading and lagging strands and the dynamics that take place on the replication fork in eukaryotes.

Here's the abstract and references.

Polymerase Dynamics at the Eukaryotic DNA Replication Fork
Peter M. J. Burgers

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

This review discusses recent insights in the roles of DNA polymerases (Pol) {delta} and {epsilon} in eukaryotic DNA replication. A growing body of evidence specifies Pol {epsilon} as the leading strand DNA polymerase and Pol {delta} as the lagging strand polymerase during undisturbed DNA replication. New evidence supporting this model comes from the use of polymerase mutants that show an asymmetric mutator phenotype for certain mispairs, allowing an unambiguous strand assignment for these enzymes. On the lagging strand, Pol {delta} corrects errors made by Pol {alpha} during Okazaki fragment initiation. During Okazaki fragment maturation, the extent of strand displacement synthesis by Pol {delta} determines whether maturation proceeds by the short or long flap processing pathway. In the more common short flap pathway, Pol {delta} coordinates with the flap endonuclease FEN1 to degrade initiator RNA, whereas in the long flap pathway, RNA removal is initiated by the Dna2 nuclease/helicase.

J. Biol. Chem., Vol. 284, Issue 7, 4041-4045, February 13, 2009



If you are interested in DNA replication, I recommend the chapter on Watson's 'Molecular Biology of the Gene' textbook. I think that chapter gives a nice intro on the concepts you need.


1 Johnson A, O'Donnell M. Cellular DNA replicases: components and dynamics at the replication fork. Annu Rev Biochem. 2005;74:283-315.

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Wednesday, January 14, 2009

Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis



Hamdan SM, Loparo JJ, Takahashi M, Richardson CC, van Oijen AM.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.

In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments. At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. Here we use single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every Okazaki fragment.

Nature 457, 336-339 (15 January 2009) | Published online 23 November 2008

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Tuesday, January 13, 2009

The replisome uses mRNA as a primer after colliding with RNA polymerase



Pomerantz RT, O'Donnell M.

The Rockefeller University, Howard Hughes Medical Institute, 1230 York Avenue, New York, New York 10021, USA.


Replication forks are impeded by DNA damage and protein-nucleic acid complexes such as transcribing RNA polymerase. For example, head-on collision of the replisome with RNA polymerase results in replication fork arrest. However, co-directional collision of the replisome with RNA polymerase has little or no effect on fork progression. Here we examine co-directional collisions between a replisome and RNA polymerase in vitro. We show that the Escherichia coli replisome uses the RNA transcript as a primer to continue leading-strand synthesis after the collision with RNA polymerase that is displaced from the DNA. This action results in a discontinuity in the leading strand, yet the replisome remains intact and bound to DNA during the entire process. These findings underscore the notable plasticity by which the replisome operates to circumvent obstacles in its path and may explain why the leading strand is synthesized discontinuously in vivo.


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Another paper by Mike O'Donnell  highlighting the complexities of the DNA replication machinery and suggesting an exciting explanation to an 'ancient' observation regarding the discontinuous synthesis not only of the lagging strand (which is explained by the antiparallel disposition of the DNA strands that must be replicated by a single advancing holoenzyme), but also of the leading strand in vivo. Indeed, Okazaki, in his original work, observed this discontinuity in vivo and has been demonstrated by a number of in vivo studies since then. 

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