Takata Laboratory
Institute for Basic Science - Center for Genomic Integrity
Introduction
We aim to understand ways to repair DNA double strand breaks (DSBs) and DNA interstrand crosslinks (ICLs). DSBs and ICLs, which are induced by multiple cancer therapies, are a particularly deleterious form of DNA damage that can result in cell death if not repaired.
Additionally, the repair process itself can result in deletions, insertions and translocations, hallmarks of genomic instability that are often found in cancer. A better understanding of how cells die as a result of DSBs and ICLs or survive with increased genomic instability will lead to improved procedures for cancer treatment and prevention.
We investigate the mechanisms of how human cells process DSBs or ICLs. This will yield a better understanding of how these processes maintain genome integrity, and how tumor cells gain resistance to cancer therapies.
Our long-term goal is to find a way to prevent cancers. One of our research topics is therefore to elucidate the mechanism of how DNA mutations accumulate during cancer development.
Keywords: DNA polymerase theta (POLQ); DNA helicase HELQ; Radiation therapy, Chemotherapy, DNA double strand breaks; High LET radiation; DNA crosslinks; CRISPR/Cas9; Next-Generation Sequencing (NGS); Cancer mutation signatures
The POLQ/ HELQ/ POLN family in DNA damage tolerance
We characterize POLQ and HELQ and their associating factors to understand ways to repair DSBs and ICLs.
In Drosophila, mutations in the mus308 locus cause hypersensitivity to ICL reagents. Its human homologs are DNA helicase HELQ, DNA polymerase θ and ν ( POLQ and POLN).
HELQ is important for tolerating ICLs, while POLQ is involved in one of the DNA double strand break (DSB) repair pathways, and POLN does not influence cellular sensitivity to ICLs or DSBs.
DNA polymerase θ (POLQ)-mediated end joining (TMEJ)
is a distinct pathway for mediating DSB repair
There are three major pathways for repair of DSBs: (i) nonhomologous end joining (NHEJ), (ii) homologous recombination (HR) and (iii) DNA polymerase θ (POLQ)-mediated end joining (TMEJ). TMEJ mediates the joining of two resected 3′ ends harboring DNA sequence microhomology (MH). TMEJ is an important alternative to the major DSB repair pathways, HR and NHEJ. The requirement of POLQ for the viability of BRCA-mutated cancer cells underscores the importance of TMEJ. The choice of DSB repair pathway influences the fidelity of DSB repair, which eventually influences the rate of tumorigenesis.
POLQ Repairs Complex DNA Double-strand Breaks
Induced by Next-generation Radiotherapy
Ionizing radiation (IR) therapy is frequently used in the treatment of cancer and is believed to destroy cancer cells by inducing DNA breaks. The newest type of radiation therapy harnesses radiation produced by a particle accelerator, which consists of charged heavy particles such as carbon ions. The particle accelerator accelerates the carbon ions to about 70% of the speed of light, which collides with and destroys the DNA of cancer cells.
These ions have a high linear energy transfer (LET) and release most of their energy within a short range, called the Bragg peak. The next-generation cancer radiotherapy works by focusing the Bragg peak on the tumor, which has the added benefit of minimizing damage to surrounding normal tissues compared to the commonly used low LET radiation such as gamma or x-rays.
(A) Depth dose distribution for low LET x-rays and monoenergetic Bragg curve for high LET carbon ions. (B) Carbon ions produce more “complex” DSBs and x-rays produce relatively “clean” DSBs.
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). This TLS activity was found to be the biologically significant factor that allows for complex DSB repair. The inhibition of POLQ may augment the efficacy of heavy ion radiation therapy.
(A) POLQ is able to anneal two single-stranded DNA tails utilizing a short homology sequence and is able to bypass DNA damage. (B) A model of POLQ-mediated repair following high LET radiation. POLQ promotes synapsis formation of the two resected 3’-single-stranded DNA tails and efficiently bypasses DNA damage located on the tails.
(A) Effect of POLQ deletion on cell survival fraction after carbon ion or x-ray irradiation. (B) POLQ deletion increases chromatid breaks after carbon ion irradiation.
HELQ operates DNA repair and signaling in response to ICL
HELQ is a 3′–5′ DNA helicase. HELQ operates in an arm of DNA repair and signaling in response to ICLs. HELQ is associated with the RAD51 paralogs RAD51B/C/D and XRCC2 (BCDX2 complex) and with the DNA checkpoint proteins ATR/ATRIP. After treatment with ICL-forming agents, phosphorylation of the ATR substrate CHK1 is reduced in HELQ knockout cells, and the checkpoint dependent accumulation of G2/M cells is attenuated.
