V. Analysis of Cfp1 Function in DNA Damage Sensitivity
5. CXXC1 -/- ES cells exhibit decreased Ape1 protein expression and endonuclease activity
The majority of DNA damaging agents that CXXC1-/- ES cells exhibit increased sensitivity to cause lesions that can be repaired by the BER pathway. Microarray analysis demonstrates that mRNA expression of genes involved in DNA repair pathways are altered in CXXC1-/- ES cells compared to CXXC1+/+ ES cells (data not shown). Interestingly, CXXC1-/- ES cells demonstrate decreased (~33%) mRNA expression of Ape1 (data not shown). Ape1 is the major apurinic/apyrimidinic endonuclease activity in human cells responsible for recognition and incision of non- coding AP sites in DNA arising as a consequence of spontaneous, chemical, or DNA glycosylase-mediated hydrolysis of the Ν-glycosyl bond initiated by the BER pathway (Raffoul 2004; Demple 1994). In addition, targeted depletion of Ape1 protein by anti- sense oligonucleotides renders mammalian cells hypersensitive to several DNA damaging agents including MMS, TMZ, cisplatin, and H2O2 (Walker, 1998; Yang, 2005; Ono, 1994). Therefore, the protein expression level and endonuclease activity of Ape1 was determined in CXXC1+/+, CXXC1-/-, and DNMT1-/- ES cells.
FIGURE 49. Ape1 protein expression and endonuclease activity in CXXC1+/+, CXXC1-/-, and DNMT1-/- ES cells.
(A) Western blot analysis was performed on protein extracts collected from CXXC1+/+, CXXC1-/-, DNMT1-/-, and CXXC1-/cDNA ES cells using antiserum directed against Cfp1, Dnmt1, Ape1, Xrcc1, and β-actin as a loading control. The graph represents results from at least three independent experiments and error bars represent standard error. Asterisk denotes a significant (p<0.05) decrease in Ape1 protein expression when compared to CXXC1+/+ ES cells.
Dr. Melissa L. Fishel. A 26 bp oligonucleotide substrate containeda single THF residue in the middle, yielding a HEX-labeled 13-mer fragment upon excision. Ape1 activity was measuredby incubating HEX-labeledexcess oligo substrate with 15.63 ng and 7.81 ngcellular protein followed by quantification of the level of the 13-mer (AP
endonuclease cleavage product) relative to the 26-mer substrate. The bottom panel is Western blot analysis for β-actin using 2.5 àg whole cell extract to show equal protein concentration of CXXC1+/+, CXXC1-/-, and DNMT1-/- ES cells using same protein extracts utilized for the Ape1 endonuclease activity assay. The graph represents results from at least three independent experiments and error bars represent standard error.
Asterisks denotes a significant (p<0.05) decrease in Ape1 endonuclease activity when compared to CXXC1+/+ ES cells.
Western blot analysis revealed ~50% decrease in Ape1 protein expression in CXXC1-/- ES cells and a slight increase in Ape1 protein expression in DNMT1-/- ES cells (Fig. 49). In addition, expression of Cfp1 in the CXXC1-/- ES cells (CXXC1-/cDNA) was sufficient to reconstitute Ape1 protein expression back to levels observed in CXXC1+/+
ES cells (Fig. 49). In contrast, protein expression of another downstream component involved in the BER pathway, Xrcc1, was unchanged between CXXC1+/+ and
CXXC1-/- ES cells (Fig. 49A). Xrcc1 functions as both a scaffold protein and modulator of BER via functional and physical interaction with Ape1, bridging the incision and nick-sealing steps of BER (Raffoul 2004). Xrcc1 interacts with DNA ligase III and DNA β-polymerase to play a role to regenerate an intact DNA strand (Kulkari 2008).
Ape1 endonuclease activity assays were performed by Dr. Melissa Fishel. Ape1
endonuclease activity was measured by incubation of protein cell extracts with a 26-mer HEX-labeled oligonucleotide substrate that contains an AP site. A 13-mer
oligonucleotide is produced upon cleavage of the AP site by Ape1 endonuclease activity. The amount of cleavage product (13-mer) relative to the amount of original substrate oligonucleotide (26-mer) is indicative of Ape1 endonuclease activity.
Consistent with Ape1 protein levels, Ape1 endonuclease activity is significantly (~40-50%) decreased in CXXC1-/- ES cells compared to CXXC1+/+ ES cells (Fig. 49B).
Immunoprecipitation was performed to determine if Ape1 interacts with Cfp1.
Full-length FLAG-epitope tagged Cfp1 was expressed in human embryonic kidney (HEK-293) cells, followed by isolation of FLAG agarose beads and detection of interacting protein by Western blot. However, Ape1 was not detected as a pull-down
FIGURE 50. Ape1 does not interact with Cfp1.
Nuclear extracts prepared from Human embryonic kidney (HEK-293) cells expressing FLAG-Cfp1 (full-length 1-656, 1-656 C580A, or 1-656 C586A) were subjected to FLAG-immunoprecipitation followed by western blot analysis. Western blots were probed with Ape1 and FLAG antibodies.
FIGURE 51. Ape1 is distributed throughout the nucleus and cytoplasm in ES cells.
Endogenous nuclear distribution of Ape1 was detected in CXXC1+/+ and CXXC1-/- ES cells using mouse anti-Ape1 antibody and bovine anti-mouse IgG- fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Nuclei were counterstained with 4,6-diamidino-2-phyenylindole (DAPI) and observed by confocal
regulated process and is quite variable, where some cell types exhibit only nuclear localization of Ape1, others display only cytoplasmic, while others show both nuclear and cytoplasmic localization of Ape1 (Tell 2005). In addition, Ape1 subcellular distribution pattern is linked to different pathological conditions, including cancer and neurodegenerative diseases (Tell 2009). Therefore, immunohistochemical staining followed by confocal microscope analysis was carried out to determine if there was a difference in the subcellular localization of Ape1 in CXXC1+/+ and CXXC1-/- ES cells.
However, there was no apparent difference in Ape1 localization in CXXC1-/- ES cells compared to CXXC1+/+ ES cells (Fig. 51). Each cell line demonstrated a punctate staining of Ape1 distributed mostly within the nucleus with very little cytoplasmic localization (Fig. 51).