Mechanisms of RNA-DNA repair

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Polymerase theta (Polq) is a unique polymerase-helicase fusion protein that performs multiple functions in DNA repair in mammalian cells. Polq was initially characterized as a translesion DNA polymerase due to its ability to perform replication opposite DNA lesions in vitro, and Polq translesion synthesis (TLS) has now been confirmed in cells. Our laboratory and others have also identified an essential role for Polq in repairing double-strand breaks (DSBs) via microhomology-mediated end-joining (MMEJ). Polq has also been implicated in replication repair, base excision repair, somatic hypermutation and class-switch recombination. Whether this unique and error- prone polymerase exhibits additional activities in DNA repair remains unknown. Here, we discover that recombinant human Polq exhibits robust reverse transcriptase (RT) activity, whereas all other human DNA polymerases tested from the A, B, Y and X families fail to perform reverse transcription beyond 1-2 nucleotides. Polq generates identical reverse transcription products as retroviral RTs. Unexpectedly, Polq exhibits a significantly higher velocity and fidelity of deoxyribonucleotide incorporation on RNA versus DNA templates. These preliminary studies identify Polq as a novel RT in mammalian cells. We plan to elucidate the molecular basis of Polq RT activity in vitro and investigate multiple RNA-templated DNA repair (RNA-DNA repair) mechanisms in cells and in vivo by developing the following Aims: 1. To characterize Polq reverse transcriptase activity in vitro; 2. To elucidate the molecular basis of Polq reverse transcriptase activity; 3. To investigate RNA-DNA repair using cellular and animal models. We anticipate that these studies will confirm and characterize Polq RT activity in vitro and in a biological setting, and elucidate novel mechanisms of mammalian RNA-DNA repair.