Geunil Yi ^1,2, Yubin Sung ¹, Chanwoo Kim ³, Jae Sun Ra ¹, Hirokazu Hirakawa ⁴, Takamitsu A Kato ⁵, Akira Fujimori ⁴, Hajin Kim ^1,3, Kei-Ichi Takata ^1,2

¹Center for Genomic Integrity, Institute for Basic Science, Ulsan 44919, Republic of Korea.
²Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea.
³Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea.
⁴Department of Charged Particle Therapy Research, Institute for Quantum Medical Science, Chiba 263-8555, Japan.
⁵Department of Environmental & Radiological Health Sciences, Colorado State University, Colorado 80523, USA.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.
To whom correspondence should be addressed. : Kei-Ichi Takata

DNA polymerase θ (POLQ) is a unique DNA polymerase that is able to perform microhomology-mediated end-joining as well as translesion synthesis (TLS) across an abasic (AP) site and thymine glycol (Tg). However, the biological significance of the TLS activity is currently unknown. Herein we provide evidence that the TLS activity of POLQ plays a critical role in repairing complex DNA double-strand breaks (DSBs) induced by high linear energy transfer (LET) radiation. Radiotherapy with high LET radiation such as carbon ions leads to more deleterious biological effects than corresponding doses of low LET radiation such as X-rays. High LET-induced DSBs are considered to be complex, carrying additional DNA damage such as AP site and Tg in close proximity to the DSB sites. However, it is not clearly understood how complex DSBs are processed in mammalian cells. We demonstrated that genetic disruption of POLQ results in an increase of chromatid breaks and enhanced cellular sensitivity following treatment with high LET radiation. At the biochemical level, POLQ was able to bypass an AP site and Tg during end-joining and was able to anneal two single-stranded DNA tails when DNA lesions were located outside the microhomology. This study offers evidence that POLQ is directly involved in the repair of complex DSBs.