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A Closer Look at DNA Replication with iPOND

By: Carol A. Rouzer, VICB Communications
Published: July 14, 2011

A new method of selectively capturing proteins involved in DNA replication and the DNA damage response promises to revolutionize our knowledge of these critical processes.

DNA replication is a complex process requiring the intricately timed interaction of a large number of enzymes and accessory binding proteins (Figure 1).  Damage encountered during replication, termed replication stress, can stall replication and activate the DNA damage response (DDR).  The DDR protects, repairs, and promotes the faithful completion of chromosome replication.  Alternatively, if the damage burden is too large and cannot be repaired, the DDR targets the damaged cells for cell death.  These DDR activities ensure that each daughter cell will be an exact and genomically intact replica of the parental cell.  Mutations in DDR proteins lead to the accumulation of genomic instability that ultimately causes human diseases such as cancer.  Therefore understanding the DDR is important to understanding mechanisms of disease etiology. The regulation of the DDR in response to insults such as irradiation and chemical toxins has been thoroughly studied; however attempts to investigate how the DDR is regulated under conditions of replication stress have been hampered by a lack of methods with sufficient sensitivity and resolution to explore the changes that occur over time at the replication fork.  Now, Vanderbilt Institute of Chemical Biology  (VICB) member David Cortez and his laboratory propose a new method, iPOND (isolation of Proteins On Nascent DNA), that provides a high resolution picture of DNA replication at both healthy and stalled forks [B. M. Sirbu et al. (2011) Genes and Development, 25, 1320].


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 Wikimedia Commons under the GNU Free Documentation License.

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. 

Figure 2
. The click chemistry reaction, and the structures of EdU and the biotin azide reagent used in these studies.

The Cortez lab realized that they could control the labeling of the replication fork and/or its surrounding regions by timing the addition of EdU to cells undergoing DNA replication.  The continuous presence of EdU labels the fork itself and a growing area behind the fork as the incubation time continues (Figure 3B).  Alternatively, a brief pulse of EdU followed by a chase with thymidine labels just the fork initially but as the chase time continues, the labeled region will be located at greater and greater distances from the fork as it progresses down the DNA helix (Figure 3C).  By applying these various pulse-chase techniques, the investigators could observe the changing patterns of proteins present during replication itself, and then later as chromatin is reconstructed around the newly synthesized DNA.  Stalling replication by adding hydroxyurea allowed them to dissect the evolution of the DDR in response to replication stress.

Figure 3. (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 and fragment the DNA.  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.  (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.

Initial studies focused on replication at active forks.  The iPOND method easily identified known DNA replication proteins, including proliferating cell nuclear antigen (PCNA), chromatin assembly factor 1 (CAF-1), and replication protein A (RPA).  The high sensitivity of the method became apparent when the data also revealed the presence of POLE2 and POLE3, two proteins that are present at levels of only one or two molecules per replication fork.

Further validation of the iPOND approach included monitoring the process of chromatin assembly, which involves the binding of histones onto newly synthesized DNA.  While brief pulses of EdU confirmed the presence of proteins involved in replication, more prolonged incubation periods revealed proteins associated with chromatin assembly, including first histones H2B and H3, and later the linker histone H1.  Consistent with prior data, the investigators found that the newly synthesized histone H4 deposited onto nascent DNA was acetylated at lysine-5 and lysine-12.  As the chromatin maturation process advanced, these acetyl groups were removed in a time-dependent manner.

The use of hydroxyurea to stall replication led to different patterns of protein recruitment and post-translational modifications at the stalled replication fork (Figure 4).  For example, an immediate drop in PCNA and CAF-1 to new steady state levels at about 20 - 30% of their values at the active fork was observed.  RPA levels remained constant, but phosphorylation of the protein was apparent at serine-33 beginning at 10 minutes and at serines-4 and -8 beginning at two hours after hydroxyurea addition.  The accumulation of phosphorylated histone H2AX (γH2AX) also began as early as 10 minutes and continued to spread outward from the stalled fork, eventually encompassing a region tens of thousands of nucleotides in length.


Figure 4. Following an EdU pulse label, hydroxyurea (yellow) is added to the cells to stall replication.  Over time, DDR response proteins (red, orange, green) assemble at the replication fork to repair damage that results from prolonged replication stress.  Figure kindly provided by Bianca Sirbu of the Cortez lab.

Although γH2AX is normally associated with the presence of double strand breaks, other DDR proteins did not appear in large quantities at the stalled fork until two to four hours after the addition of hydroxyurea.  The presence of these proteins, including MRE11, Ku70, Ku80, and Rad51, suggests that strand breakage begins to occur after about two hours of blocked replication.  Inhibitor studies suggested that the protein kinase ATR, which mediates the early phosphorylation of RPA at serine-33, also mediates the initial phosphorylation and spreading of histone γH2AX.  The kinases ATM and DNA-PK appear to play a role in the latter phosphorylations of RPA (serines-4 and -8) and γH2AX.

The data provide a strikingly clear picture of the spatial and temporal dynamics of proteins involved in DNA replication and in the response to replication stress.  These initial studies validate iPOND as a valuable new technique for the study of these critical processes.  In this work, the investigators used western blot analysis to identify individual proteins and assess their post-translational modification.  Future plans to employ mass spectrometry for protein analysis should yield even greater accuracy and sensitivity with this exciting new approach.















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