UNDERSTANDING THE ROLE OF POLQ IN CHROMOSOMAL DNA REPLICATION UNDER STRESSFUL CONDITIONS

DNA polymerase-theta (Polq) is a class A family DNA polymerase comprising of a helicase-like domain on the N-terminus and a DNA polymerase domain on the C-terminus. Pol-theta overexpression in breast and ovarian cancer patients correlate with high tumor grade and poor response to chemotherapeutic drugs. Consistently, Pol-theta inhibition is synthetically lethal with inactivation of BRCA1/2 genes, which are often found mutated in these tumors. The ability of Pol-theta to sustain viability of BRCA1/2 defective cancer cells has been attributed to its role in alternative end-joining repair of double strand breaks (DSBs) resulting from collapsed replication forks. In addition to DSBs a major role in BRCA1/2 activity is the suppression of defects associated with faulty DNA replication. Indeed, the occurrence of extensive DNA replication defects ranging from single stranded DNA gap accumulation to nascent DNA degradation in the absence of BRCA1/2 and RAD51 has been previously demonstrated. However, the role of Pol-theta in counteracting defective DNA replication in the absence of functional BRCA1/2 and RAD51 is poorly understood. To address this question, we cloned and purified the full length and different domains of Xenopus laevis Pol-theta and generated antibody to study Pol-theta function in replicating Xenopus egg extracts. Our preliminary findings indicate that Pol-theta has replication dependent and independent functions. Significantly, although dispensable for normal chromosomal DNA replication, Pol-theta is strongly enriched at stalled forks upon replication stress conditions induced by aphidicolin. Using DNA electron microscopy, we discovered that Pol-theta overexpression suppresses ssDNA gaps at the replication fork and replication fork reversal triggered by aphidicolin-induced fork stalling. Therefore, our results suggest that Pol-theta not only repairs DSBs but also prevents the occurrence of potentially harmful DNA replication intermediates. We are currently investigating Pol-theta function in relation to replicative defects arising in the absence of BRCA1/2 and RAD51. Better understanding of Pol-theta function at stalled forks will help to target breast and ovarian cancers more effectively.