DNA Replication Fork - McGraw Hill Education

Moyer SE, Lewis PW and Botchan MR (2006) Isolation of the Cdc45/Mcm2‐7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proceedings of the National Academy of Sciences of the USA 103(27): 10236–10241.

DNA Synthesis | Dna Replication | Telomere

Four enzymes are included in this model of the DNA replication fork process:

DNA Replication 3-D Animation from DNA ..

Chastain PD, Makhov AM 2nd, Nossal NG and Griffith J (2003) Architecture of the replication complex and DNA loops at the fork generated by the bacteriophage t4 proteins. Journal of Biological Chemistry 278(23): 21276–21285.

The DNA near each replication fork is coated with single-stranded ..

A comprehensive proteomics analysis of proteins at normal and stressed replication forks illuminates the role of ATR in preventing fork collapse.

Replication fork: The site of DNA replication where two replicating single‐stranded DNA separates.

where separation of the DNA occurs is called the replication fork

The key to iPOND is the use of “click” chemistry, a copper-catalyzed reaction between an azide and an alkyne, that has been employed extensively to label and retrieve biomolecules from the complex cellular environment (Figure 2). The Cortez lab took advantage of the fact that cells readily incorporate the alkyne-containing nucleotide 5′-ethynyl-2′-deoxyuridine (EdU) in place of thymidine as they replicate DNA. Reaction of DNA containing EdU with a biotin azide reagent (designed and synthesized in the lab of VICB member Ned Porter) under click chemistry conditions attaches a biotin label to the alkyne moiety of the incorporated EdU in the DNA. Following fragmentation of the DNA, biotin-labeled DNA fragments are captured on streptavidin-coated beads, providing a rapid and efficient method for purification (Figure 3A). The use of formaldehyde to covalently cross-link DNA to any associated proteins provides a means to isolate and identify proteins bound to the DNA at the site of the EdU-biotin label.

Replication Site where DNA synthesis ..

Bambara RA, Murante RS and Henricksen LA (1997) Enzymes and reactions at the eukaryotic DNA replication fork. Journal of Biological Chemistry 272: 4647–4650.

replication fork; Okazaki fragment; DNA polymerases; primer RNA; replication restart

DNA replication is the process of ..

Bouche JP, Rowen L and Kornberg A (1978) The RNA primer synthesised by primase to initiate phage G4 DNA replication. Journal of Biological Chemistry 253: 765–679.

Your goal is to speed up the DNA replication process that occurs in every cell in every living creature as part of mitosis or meiosis.

The E. coli DNA Replication Fork | Read by QxMD


Figure 2. (A) Diagram illustrating the iPOND method. 1. Newly synthesized DNA incorporates EdU at the replication fork. 2. Formaldehyde is used to cross-link the DNA with any proteins associated at the fork region. 3. The cells are permeabilized, and reaction with the biotin azide reagent under click chemistry conditions attaches biotin to EdU in the DNA. 4. The cells are lysed and sonicated to release the DNA and break it into fragments. 5. DNA fragments are isolated by attachment to streptavidin-coated beads. 6. Following elution from the beads, the formaldehyde cross-links are reversed by high temperature incubation, releasing the proteins for analysis by western blot as shown in the figure or by mass spectrometry as carried out in the current study. (B) The region of DNA labeled with EdU (gold) starts at the replication fork and grows longer as the fork progresses along the DNA helix. With prolonged EdU incubation, the regions labeled initially undergo post-replication processes, such as chromatin assembly, and the associated proteins change as the process progresses. Newly synthesized DNA is associated with replication proteins such as PCNA and CAF-1. Over time, histones bind to the DNA in the process of chromatin assembly. (C) Incubation with a brief pulse of EdU followed by a chase of thymidine leads to EdU incorporation in a small segment of DNA that becomes more distant from the moving replication fork as the chase time increases. This allows the selective investigation of proteins at different stages of replication and post-replication processing. Figure kindly provided by Bianca Sirbu of the Cortez lab.

Primosome: A name given to the protein complex capable of duplex DNA unwinding and primer RNA synthesis at the replication fork.

The DNA near each replication fork is ..

Figure 1. DNA replication is a complex process. The two strands of the double helix are unwound by the action of topoisomerase and helicase to form the replication fork. The single strands are stabilized by binding proteins, and the two strands are replicated in reverse directions, beginning with an RNA primer. This diagram shows the general structure of the replication fork as the process progresses down the DNA strand, but it includes only a fraction of the large number of proteins involved. Figure reproduced from under the .