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Protecting Cells Against ssDNA Abasic Site-Mediated Mutations

 

By: Carol A. Rouzer, VICB Communications
Published: December 14, 2018

 

HMCES binds tightly to abasic sites in single-stranded DNA in a way that protects against error-prone processing.

 

Abasic sites (apurinic/apyrimidinic or AP sites) are among the most common forms of DNA damage. They can occur through spontaneous hydrolysis of the glycosidic bond joining the purine or pyrimidine base to the deoxyribose ring (a reaction that is more common following formation of certain kinds of DNA adducts), or they can result from the action of DNA glycosylases that remove damaged bases as the first step of the base excision repair pathway. When present in double-stranded DNA, AP sites can readily be repaired by removal of the damaged nucleotide and then using the remaining undamaged strand as a template for filling in the gap. However, AP sites can also occur in single-stranded DNA (ssDNA). In this case, no template is available for repair, and the process is frequently carried out by translesion polymerases that exhibit poor fidelity and frequently lead to mutations. Alternatively, AP endonucleases may remove an AP site in ssDNA leading to a double strand break, another outcome that often can lead to inaccurate repair and permanent DNA damage. Relatively little is known about the ways in which the cell protects itself from AP sites in ssDNA, so the new discovery from Vanderbilt Institute of Chemical Biology member David Cortez and his laboratory that the protein HMCES protects ssDNA AP sites in replicating DNA is of particularly high importance [K. N. Mohni, et al., (2018) Cell, published online December 13, DOI: 10.1016/j.cell.2018.10.055].


The researchers first became interested in HMCES (5-hydroxymethylcytosine-binding, embryonic stem cell-specific) as a result of studies conducted with the iPOND (isolation of proteins on nascent DNA, Figure 1) method. iPOND identifies proteins associated with DNA that is undergoing replication. HMCES was among those proteins, a somewhat unexpected finding as this protein was initially discovered through its ability to bind to 5-hydroxymethylcytosine in embryonic stem cells. This original finding suggested a role for HMCES in epigenetic control of DNA homeostasis; however, a protein similar to HMCES is present in nearly all organisms, even those that do not form 5-methylcytosine or 5-hydroxymethylcytosine. Furthermore, in bacteria, the homologous protein is associated with the DNA damage response, suggesting a potential role for HMCES in DNA repair. A notable feature of all these proteins is the presence of an SOS (DNA damage) response-associated peptidase (SRAP) domain, characterized by a conserved cysteine at position 2 and a catalytic pocket that also contains a conserved glutamate and histidine residue. This catalytic pocket is surrounded by a positively charged surface through which it may interact with DNA (Figure 2). Thus, the researchers decided to test the hypothesis that HMCES is involved in some aspect of DNA replication and/or repair.

 

 

 

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. Diagrammatic representation of the surface of the SRAP domain of HMCES colored to indicate the presence of positive charge (blue) and negative charge (red). The catalytic pocket and the basic surface are indicated by arrows. Created using UCSF Chimera software from PDB file number 5KO9.

 

 

 

The investigators began their studies by creating lines of U2OS cells bearing a deletion mutation in the gene encoding HMCES. They found that these cells were more sensitive than wild-type U2OS cells to exposure to inhibitors of the ATR checkpoint kinase. Expression of HMCES in wild-type cells was higher during S phase than during phases of the cell cycle in which DNA synthesis did not occur, and the protein was observed to translocate to the nucleus following exposure to DNA damaging agents. All of these findings were consistent with a role for HMCES in DNA replication/repair.
     

Next, the researchers conducted DNA binding studies. These experiments revealed that HMCES binds strongly to ssDNA. Further investigations using site-directed mutants of HMCES demonstrated the requirement for two arginine residues in the basic surface of the SRAP domain in order for DNA binding to occur. In addition, the investigators found that HMCES interacts with PCNA (proliferating cell nuclear antigen), a protein that plays an important role in DNA replication. Consistently, HMCES contains a PCNA-interacting peptide (PIP) box, a motif found in the majority of proteins that interact with PCNA. Mutation of a key residue within this motif eliminated PCNA binding.
     

U2OS cells deficient in HMCES exhibited no change in 5-methylcytosine or 5-hydroxymethylcytosine levels; however, the investigators identified 22 genes that were either up- or down-regulated in these cells. Many of these genes were associated with the DNA damage or replication stress responses. These findings further supported the hypothesis that HMCES is more likely involved in DNA replication/repair than epigenetic modulation. Consistently, HMCES-deficient cells were hypersensitive to both infrared and ultraviolet radiation and to methyl methanesulfonate. All of these are DNA damaging agents, each of which induces a different kind of damage leading to repair by a distinct mechanism, but they all have in common the ability to induce AP site formation. In contrast, HMCES-deficient cells did not exhibit increased sensitivity to DNA damaging agents that do not induce AP sites. This led the investigators to hypothesize that HMCES is involved in the response to AP sites. Their hypothesis was further supported by the finding that DNA from HMCES-deficient cells contained higher levels of AP sites than that of wild-type cells.
     

The researchers next found that the SRAP domain from HMCES binds ssDNA containing an AP site with extremely high affinity. The interaction was stable to protein denaturation, suggesting a covalent linkage. Mutation studies demonstrated that both the active site cysteine and basic surface arginine residues were required for this association. The deoxyribose sugar in AP sites exists in an equilibrium between the ring-closed deoxyfuranose configuration and a ring-opened aldehyde-containing form. The HMCES SRAP domain failed to form a covalent linkage with a stable ring-closed AP site analog, suggesting that the aldehyde in the ring-opened form of the lesion reacts with the SRAP domain – likely with the conserved cysteine residue.
     

Treatment of cells with a DNA damaging agent led to increased formation of cross-links between DNA and the SRAP domain of HMCES concomitantly with a depletion of free HMCES. Levels of the DNA-HMCES cross-links were higher if the cells were treated with an inhibitor of the proteasome, suggesting that cross-link formation leads to proteasome-mediated degradation of HMCES. Consistently, exposure of cells to DNA damaging agents led to ubiquitination of HMCES, marking it for proteasomal degradation.
     

The investigators showed that HMCES can bind to AP sites in ssDNA configured as it would be found in stalled replication forks. Binding of HMCES to the AP site prevented cleavage of the site by AP endonucleases, thereby preventing the formation of a double-strand break in the DNA. Consistently, double strand breaks accumulated in cells deficient in HMCES.
     

Using SILAC (stable isotope labeling with amino acids in cell culture, Figure 3) combined with iPOND to quantify changes in proteins associated with the replication fork in HMCES-deficient versus wild-type cells, the researchers found higher levels of translesion bypass polymerases in the forks of the deficient cells. This finding suggested that translesion synthesis was helping the cells to compensate for the loss of HMCES, a conclusion that was supported by the observation that siRNA-mediated knockdown of the translesion polymerase REV3 in HMCES-deficient cells resulted in a marked loss in cell viability, particularly after exposure to ultraviolet light. The researchers also demonstrated the accumulation of more mutations in the DNA of HMCES-deficient cells than wild-type cells following UV exposure. These results suggested that use of translesion synthesis by cells to overcome HMCES deficiency leads to an excess of mutations.

 

 

     

FIGURE 3. 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.

 

 

Together the results suggest that the main function of HMCES is to bind to and protect AP sites in ssDNA until repair can be accomplished. The stabilization occurs via covalent binding of HMCES to the AP site, leading ultimately to the degradation of the HMCES. Hence, it acts as a suicide enzyme. It is not completely clear how HMCES mediates repair of AP sites, but the data suggest that it helps to prevent the use of translesion polymerases or the formation of double strand breaks, both of which lead to high mutation rates (Figure 4).

 

 

FIGURE 4. Proposed mechanism for the action of HMCES (blue) at the replication fork. HMCES binds to an AP site (yellow) on single-stranded DNA while also interacting with PCNA. Binding of HMCES prevents the action of translesion polymerases (green) that would likely lead to error-prone repair, and it also blocks the action of AP endonucleases (red) that create double strand breaks after they remove an AP site.

will better define its epitope and explore its potential therapeutic use in humans.

 

 

 

View Cell article: HMCES Maintains Genome Integrity by Shielding Abasic Sites in Single-Strand DNA

 

 

 

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