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A Newly Discovered Protein Helps to Maintain Genome Integrity

 


By: Carol A. Rouzer, VICB Communications
Published:  July 24, 2017

 

RADX binds to single-stranded DNA and opposes the function of RAD51 to maintain replication stability.

 

 

Single-stranded DNA (ssDNA) that is formed during the course of DNA replication or repair and at telomeres must be protected from degradation by endonucleases. To this end, the cell has multiple ssDNA-binding proteins that both protect the DNA and promote necessary metabolic processes. Among these are replication protein A (RPA), which associates with ssDNA at the replication fork to facilitate lagging-strand synthesis and prevent fork collapse, and RAD51, which forms filaments on ssDNA at resected double strand breaks to promote homology-directed repair (HDR). RAD51 also promotes reversal of stalled replication forks and prevents the degradation of newly synthesized DNA strands. Now, Vanderbilt Institute of Chemical Biology members David Cortez and Walter Chazin with their laboratories report a new protein, RADX (RPA-related RAD51-antagonist on X-chromosome), that works to block RAD51 activity. Their finding has important implications for DNA replication and repair and provides new insights into the resistance of some forms of cancer to chemotherapy [H. Dungrawala, K. Bhat, et al., (2017) Mol. Cell, published online July 20, DOI: 10.1016/j.molcel.2017.06.023].

 

The discovery of RADX grew out of earlier experiments using iPOND (identification of Proteins on Nascent DNA), a method devised in the Cortez lab to capture and identify proteins associated with replicating DNA (Figure 1). An expansion of this approach to include SILAC (Stable Isotope Labeling with Amino acids in Cell culture, Figure 2) enabled the investigators to quantify changes in proteins that occur at the replication fork in response to stress induced by hydroxyurea. The results of these experiments demonstrated that the amounts of most chromatin proteins, such as histones, were not affected by hydroxyurea treatment. In contrast, proteins required for the replication process decreased over time following addition of hydroxyurea, as ongoing replication terminated and new origins failed to fire. In addition, proteins associated with the DNA damage response accumulated at the fork as the result of hydroxyurea-induced stress. Of these, the protein encoded by open reading frame 57 on the X chromosome was particularly abundant. Subsequent structure and function studies of this protein led the investigators to name it RADX.

 

 

 

 

FIGURE 1. Figure 1. (A) Diagram illustrating the iPOND method.  1. Newly synthesized DNA incorporates ethinyl-deoxyuridine (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 a 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 or mass spectrometry.  (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.

 

 

FIGURE 2. SILAC method for protein quantitation. (A) Cells are grown in the presence of lysine and arginine uniformly labeled with 12C and 14N or 13C and 15N to produce "light" and "heavy" cells, respectively. The cells are grown with the labeled amino acids until all cellular proteins contain those amino acids. In this example, the heavy cells are then exposed to the desired treatment while the light cells serve as controls. The cells are lysed, and protein extracts are prepared and quantified. The heavy and light extracts are then combined in equal quantities of protein and subjected to proteomics analysis by mass spectrometry. (B) The mass spectrometer can detect the difference between the masses of lysine- or arginine-containing peptides derived from each heavy- and light-labeled protein. In this example, the peptide contains one lysine residue and no arginine residues. Thus, the mass of the heavy peptide is 8 Da greater than that of the light peptide. If the signals from the heavy and light peptides are the same (center), then the treatment had no effect on the levels of this protein. If the signal from the light protein is larger (left), then the treatment reduced the levels of this protein, while if the signal of the heavy protein is larger (right), the treatment increased the levels of the protein, relative to those in the control.

 

 

To learn more about RADX's function the researchers used siRNA to knockdown expression of its gene and CRISPER-Cas9 to create a stable cell line in which the gene had been knocked out. Subsequent analysis of these cells indicated that absence of RADX resulted in an increase in DNA damage, as indicated by elevated levels of the damage marker γH2AX and an accumulation of double strand breaks as observed using a comet assay. When the investigators used siRNA to deplete the endonuclease MUS81 in RADX-deficient cells, the amount of DNA damage decreased, suggesting that RADX may help to protect ssDNA from MUS81-mediated degradation. Single molecule analysis of replicated DNA fibers (Figure 3) demonstrated that fork elongation rates were decreased in RADX-depleted cells, and an increased incidence of asymmetric sister replication forks was consistent with fork collapse in these cells.

 

 

 

FIGURE 3. Method for single strand DNA analysis. Cells are incubated for a desired time period (here 15 min) with iodo-deoxyuridine, which is incorporated into newly synthesized DNA (red). Then the cells are incubated with chloro-deoxyuridine for an equal time period to label a second stretch of DNA (green). The cells are lysed, and the DNA is denatured and incubated with antibodies that specifically bind to either iodo- or chloro-deoxyuridine. Then, secondary antibodies, each labeled with a different fluorescent tag, are bound to the first antibodies. The DNA strands are then spread onto a microscope slide, and the relative amounts of red versus green label indicates the rate of progression of the fork during each time period.

 

 

The researchers next endeavored to learn what they could about RADX from its structure. Creating a model of the protein based on its amino acid sequence, they identified five structured domains in RADX. Three of these were OB folds, known ssDNA binding domains similar to those found in RPA. In fact, the overall structure of RADX was reminiscent of the structure of RPA70, the largest RPA subunit. This finding led to the hypothesis that RADX is an ssDNA binding protein, and subsequent experiments using Flag-tagged recombinant RADX confirmed that the protein binds tightly to ssDNA. Using its homology to RPA as a guide, the investigators designed a mutant form of RADX, OBM RADX, that exhibits reduced ssDNA binding affinity. They then showed that expression of RADX but not OBM RADX reduces the number of DNA double strand breaks in RADX deficient cells, confirming that its ssDNA binding activity is important to RADX's function.


Using iPOND-SILAC, the researchers next explored proteins that are enriched at replication forks in RADX-deficient cells. They found that the most highly enriched protein was RAD51. They then used quantitative immunofluorescence to confirm this result and to show that the observed accumulation of RAD51 could be reversed by expression of RADX but not OBM RADX, suggesting that the two proteins compete for binding to ssDNA.


These findings led to the hypothesis that RADX functions to oppose the actions of RAD51, and in its absence, RAD51 hyperactivity leads to the toxic effects observed. To test this hypothesis, the investigators used siRNA to reduce RAD51 expression in RADX-deficient cells. This treatment reversed the replication fork elongation defect and accumulation of double strand breaks observed with RADX deficiency. The researchers hypothesized that, of all RAD51 functions, the one most likely to be deleterious if unopposed was fork reversal, a function that normally helps to restore stalled replication forks, but if excessive could lead to fork collapse. Consistent with this idea, they found that siRNA-mediated reduction in the expression of SMARCAL1 and ZRANB3, two other fork reversal enzymes, helped to reduce double strand breaks in RADX-deficient cells. These findings suggested that a primary function of RADX is to suppress excessive fork reversal.


Some functions of RAD51 depend on a cooperation with BRCA2, which facilitates the displacement of RPA by RAD51 on ssDNA. Cells lacking in BRCA2, a common finding in some cancers, are particularly susceptible to DNA damage due to a loss of RAD51 function. This led the investigators to hypothesize that the effects of BRCA2 deficiency might be alleviated by reducing the expression of RADX. Subsequent experiments supported this hypothesis, showing that siRNA-mediated knockdown of RADX expression prevented nascent DNA strand degradation normally observed in BRCA2-deficient cells. However, RADX depletion had no effect on the reduced ability of BRCA2-deficient cells to undergo HDR, as indicated by expression of green fluorescent protein from a gene construct that required HDR in order for expression to occur. Finally, depletion of RADX in cells deficient in either BRCA2 or RAD51 reversed, at least in part, the cells' increased susceptibility to DNA damaging agents, including several used in cancer chemotherapy.


The researchers noted that cancers bearing BRCA2 mutations are generally highly susceptible to chemotherapy at first, but often quickly acquire resistance. A common mechanism for this is the acquisition of a second BRCA2 mutation that results in a functional protein. However, the findings reported here indicate that inactivation of RADX could be another resistance mechanism in these cells. Of broader significance, these findings demonstrate the importance of a balance of RAD51 and RADX to control the processing of ssDNA at replication forks. RADX apparently accomplishes this important function without interfering with RAD51's role in HDR, thus enabling the cell to optimally maintain genomic integrity.

 

 

View Molecular Cell article: RADX Promotes Genome Stability and Modulates Chemosensitivity by Regulating RAD51 at Replication Forks

 

 

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