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Novel role of hSSB2 in the Base Excision Repair Pathway (BER) (Conference Abstract)

Background: The base excision repair (BER) pathway is responsible for removing damaged or incorrectly incorporated uracil bases in the genome. Mismatched bases that persist in the genome and remain unrepaired may result in either lethal mutations or cytotoxic DNA double strand breaks. Previous studies have determined that hSSB1 is critical for the detection, signaling and repair of cytotoxic double strand DNA breaks and oxidized DNA lesions within the genome. The role of hSSB2 is, however, less clear. In this study, we have identified that the single stranded DNA binding proteins, hSSB1 and 2, are involved in the detection and removal of uracils within the genome and function as part of the BER pathway. Method: We identified a novel role for hSSB1 and hSSB2 in BER. EMSA and incision biochemical assays were used to determine the ability of hSSB1/2 to bind uracil containing mismatches. Incision assays were used to determine the effect hSSB2 and hSSB1 have on UNG2 activity. Two cytotoxic drugs (5-fluorouracil and pemetrexed), which induce uracil misincorporation in the genome, were used to determine the cell sensitivity in control and hSSB1/2-depleted cells using a live and dead cell assay. Immunoprecipitation, immunofluorescence and Protein-Protein interactions were carried out to determine whether hSSB2 and hSSB1 interacts with key regulatory proteins of the BER pathway. Result: This study demonstrates that hSSB1 and hSSB2 proteins can recognize and bind to double stranded DNA substrates containing a uracil mismatch. Interestingly, we have identified that hSSB1 and hSSB2 have a differential preference for uracil mismatches, with hSSB1 preferentially binding UA and hSSB2 UG mismatches. Furthermore, hSSB2 induces the incision activity of UNG2 by approximately two fold for a U:G mismatch but not a U:A mismatch. A549 lung adenocarcinoma cells depleted of both hSSB1 and hSSB2 are hypersensitive to 5-fluorouracil and pemetrexed. Loss of either hSSB1 or hSSB2 alone by siRNA results in a compensatory upregulation of hSSB2 or hSSB1 respectively, suggesting over-lapping functionality and substrate specificity. Conclusion: This study highlights the importance of hSSB2 and hSSB1 in the removal of uracil from the genome. Currently, pemetrexed and fluorouracil based agents are in use for treating lung cancer. This study raises the possibility that hSSB2 and hSSB1 may be biological indicators of response to fluorouracil and pemetrexed. Further, it may be possible to develop future hSSB2/hSSB1 inhibitors that could enhance the activity of these agents in the treatment of lung cancer.