DNA primase acts as a molecular brake in DNA replication

  title={DNA primase acts as a molecular brake in DNA replication},
  author={Jong-Bong Lee and Richard K. Hite and Samir M. Hamdan and X. Sunney Xie and Charles C. Richardson and Antoine M. van Oijen},
A hallmark feature of DNA replication is the coordination between the continuous polymerization of nucleotides on the leading strand and the discontinuous synthesis of DNA on the lagging strand. [] Key Result We observe the synthesis of primers on the lagging strand to cause transient pausing of the highly processive leading-strand synthesis. In the presence of both leading- and lagging-strand synthesis, we observe the formation and release of a replication loop on the lagging strand. Before loop formation…

Coordinating DNA replication by means of priming loop and differential synthesis rate

These findings provide three synergistic mechanisms of coordination: first, primers are made concomitantly with DNA synthesis; second, the priming loop ensures efficient primer use and hand-off to the polymerase; and third, the lagging-strand polymerase copies DNA faster, which allows it to keep up with leading-Strand DNA synthesis overall.

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

Single-molecule techniques are used to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication and reveal that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release.

Timing, Coordination, and Rhythm: Acrobatics at the DNA Replication Fork*

Recently developments in single-molecule techniques have enabled the direct observation of these processes and have greatly contributed to a better understanding of the dynamic nature of the replication fork.

Coordinated DNA Replication by the Bacteriophage T4 Replisome

The T4 bacteriophage encodes eight proteins, which are sufficient to carry out coordinated leading and lagging strand DNA synthesis, and the formation of a holoenzyme complex composed of the polymerase and a processivity clamp is studied.

E. coli DNA replication in the absence of free β clamps

It is shown that replication complexes pre‐assembled on DNA support synthesis of multiple Okazaki fragments in the absence of excess β clamps, expanding the understanding of lagging‐strand synthesis and emphasizing the stability of the replisome to continue synthesis without new clamps.

Motors, switches, and contacts in the replisome.

The replisome of bacteriophage T7 contains a minimum of proteins, thus facilitating its study, and this review describes the molecular motors and coordination of their activities, with emphasis on the T7 replisomes.

Discrete interactions between bacteriophage T7 primase-helicase and DNA polymerase drive the formation of a priming complex containing two copies of DNA polymerase.

Results indicate that the T7 primase-helicase specifically engages two copies of DNA polymerase, which would allow the coordination of leading and lagging strand synthesis at a replication fork.

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery

This study uses single-molecule fluorescence methods to visualize the dynamics with which individual DNA polymerases, the replication protein responsible for DNA synthesis, associate with and dissociate from the replication machinery, and suggests that lagging-strand polymerases are exchanged at a frequency similar to that of Okazaki fragment synthesis.

Single-molecule studies of fork dynamics in Escherichia coli DNA replication

Modulation of DnaB helicase activity through the interaction with DnaG suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-Strand synthesis during slow primer synthesis on the lagging strand.

Single-molecule studies of fork dynamics in Escherichia coli DNA replication

Modulation of DnaB helicase activity through the interaction with DnaG suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-Strand synthesis during slow primer synthesis on the lagging strand.



Replisome-mediated DNA replication.

This review summarizes and compares and contrasts for these three systems the types, timing, and mechanism of reactions and of protein-protein interactions required to initiate, control, and coordinate the synthesis of the leading and lagging strands at a DNA replication fork and comments on their generality.

Lagging strand synthesis in coordinated DNA synthesis by bacteriophage t7 replication proteins.

The proteins of bacteriophage T7 DNA replication mediate coordinated leading and lagging strand synthesis on a minicircle template containing double and single-stranded DNA with a combined average length of 2600 nucleotides suggesting that the replication loop dictates the frequency of initiation of Okazaki fragments.

The Interaction between Helicase and Primase Sets the Replication Fork Clock*

The synthesis of an Okazaki fragment occurs once every 1-2 s at the Escherichia coli replication fork and requires precise coordination of the enzymatic activities required. We have shown previously

Mechanism of calf thymus DNA primase: slow initiation, rapid polymerization, and intelligent termination.

The mechanism by which calf thymus DNA primase synthesizes RNA primers was examined and the ability of primase to distinguish between primer at least 7 nucleotides long and shorter products therefore likely reflects the fact that pol alpha only utilizes primers at least7 nucleotide long.

DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase

It is reported that T7 DNA polymerase can increase the unwinding rate to 114 base pairs per second, bringing the helicase up to similar speeds compared to its translocation along ssDNA.

Mechanism and Stoichiometry of Interaction of DnaG Primase with DnaB Helicase of Escherichia coli in RNA Primer Synthesis*

It is demonstrated that the primase-helicase complex of E. coli is comprised of three molecules of primase bound to one DnaB hexamer, and it is determined that ssDNA binding activity of the DnB helicase is necessary for directing thePrimase to the initiator trinucleotide and synthesis of 11-20-nucleotide long primers.

Single-molecule studies of the effect of template tension on T7 DNA polymerase activity

Estimates of the mechanical and entropic work done by the enzyme show that T7 DNA polymerase organizes two template bases in the polymerization site during each catalytic cycle, and finds a force-induced 100-fold increase in exonucleolysis above 40 pN.

Replication by a single DNA polymerase of a stretched single-stranded DNA.

The replication rate follows an Arrhenius law and indicates that multiple bases on the template strand are involved in the rate-limiting step of each cycle, consistent with the induced-fit mechanism for error detection during replication.

The Oligomeric T4 Primase Is the Functional Form during Replication*

Results provide strong evidence for the functional oligomerization of gp61, and Mutant primases defective in either the N- or C-terminal domains and catalytically inactive could be mixed to create oligomeric primases with restored catalytic activity suggesting an active site shared between subunits.