The human DNA helicase HelQ is recruited at R-loops and helps in their resolution and repair.

Introduction mRNA-DNA R-loops created during transcription by stalling RNA polymerases pose a considerable obstacle to DNA replication and need to be cleared off for a smooth progression into the S phase of the cell cycle when the genome is replicated. Otherwise, the accumulation of R-loops can lead to genome instability. In a recent paper, Ruzov and collaborators discovered that N6-methyladenosine (m6A)-containing R-loops tend to accumulate during the G2/M phase and are depleted during the G0/G1 phase of the cell cycle. Furthermore, they showed that the m6A-binding protein YTHDF2 also associates with R-loops and a ydhdf2 knockout leads to increased R-loops levels, cell growth retardation and accumulation of double strand DNA breaks in mammalian cells. The Soultanas and Bolt groups have been working collaboratively with a human DNA repair helicase known as HelQ. HelQ is an important DNA repair protein, highlighted by multiple effects of helq gene loss, including elevated mutation rates, increased cancer incidence and sterility. Genetic analyses have led to models implicating HelQ in controlling and limiting homologous recombination when DNA replication is being repaired, so that replication can resume without genetic rearrangements occurring. But biochemical mechanisms for how HelQ helicase contributes to these events are unknown. HelQ is an ATP-dependent DNA helicase that translocates single stranded DNA (ssDNA) with 3' to 5' polarity. Recent collaborative work in the Soultanas and Bolt labs has resulted in the exciting discovery that HelQ appears to localize at R-loops, suggesting that HelQ may also be involved in processing and removing R-loops, clearing the way for replication to occur unimpeded. Hypothesis We hypothesize that problematic R-loops created by stalled transcription during the G2/M phase of the cell cycle are marked for removal during the G0/G1 clearing the genome from obstacles that might interfere with DNA replication during the S phase. HelQ is recruited at these R-loops to unwind them and clear them off. Project The main aim of this project is to provide in vivo and in vitro evidence in support of our hypothesis. 1. We will carry out immunostaining experiments with appropriate cell lines, wildtype and helq-knockout, to confirm and provide additional in vivo data that HelQ co-localizes at R-loops and also at m6A-marked R-loops 2. We will carry out immunostaining of m6A on WT and HELQ KO cells in the presence of no RNases, in the presence of RNase A only, and in the presence of both RNase A +H. This may allow us to discern whether the distribution and levels of m6A modified R-loops is altered following HELQ depletion. 3. We will use the gammaH2AX double strand marker to carry out immunostaining experiments to assess whether depletion of HelQ leads to increased DNA damage. 4. We will carry out complementation experiments to assess whether expression of HelQ rescues HelQ-depleted cells. 5. We will investigate whether HelQ co-immunoprecipitates with RNA polymerase. 6. We will express/purify the human HelQ protein and use different synthetic oligonucleotide substrates to study its activities. 7. We will carry out bio-ID experiments to identify HelQ-interactants.