Polθ promotes the repair of 5'-DNA-protein crosslinks by microhomology-mediated end-joining

Cell Rep. 2021 Mar 9;34(10):108820. doi: 10.1016/j.celrep.2021.108820.


DNA polymerase θ (Polθ) confers resistance to chemotherapy agents that cause DNA-protein crosslinks (DPCs) at double-strand breaks (DSBs), such as topoisomerase inhibitors. This suggests Polθ might facilitate DPC repair by microhomology-mediated end-joining (MMEJ). Here, we investigate Polθ repair of DSBs carrying DPCs by monitoring MMEJ in Xenopus egg extracts. MMEJ in extracts is dependent on Polθ, exhibits the MMEJ repair signature, and efficiently repairs 5' terminal DPCs independently of non-homologous end-joining and the replisome. We demonstrate that Polθ promotes the repair of 5' terminal DPCs in mammalian cells by using an MMEJ reporter and find that Polθ confers resistance to formaldehyde in addition to topoisomerase inhibitors. Dual deficiency in Polθ and tyrosyl-DNA phosphodiesterase 2 (TDP2) causes severe cellular sensitivity to etoposide, which demonstrates MMEJ as an independent DPC repair pathway. These studies recapitulate MMEJ in vitro and elucidate how Polθ confers resistance to etoposide.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Line
  • Cross-Linking Reagents / pharmacology*
  • DNA / chemistry
  • DNA End-Joining Repair / drug effects*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • DNA-Directed DNA Polymerase / deficiency
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / metabolism
  • Formaldehyde / pharmacology
  • Humans
  • Mice
  • Ovum / metabolism
  • Phosphoric Diester Hydrolases / genetics
  • Phosphoric Diester Hydrolases / metabolism
  • RNA, Guide, Kinetoplastida / metabolism
  • Xenopus / growth & development
  • Xenopus / metabolism


  • Cross-Linking Reagents
  • DNA-Binding Proteins
  • RNA, Guide
  • Formaldehyde
  • DNA
  • DNA polymerase theta
  • DNA-Directed DNA Polymerase
  • Phosphoric Diester Hydrolases
  • TDP2 protein, mouse