Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase θ

Abstract

Microhomology-mediated end-joining (MMEJ) is an error-prone alternative double-strand break repair pathway that utilizes sequence microhomology to recombine broken DNA. Although MMEJ is implicated in cancer development, the mechanism of this pathway is unknown. We demonstrate that purified human DNA polymerase θ (Polθ) performs MMEJ of DNA containing 3’ single-strand DNA overhangs with two or more base-pairs of homology, including DNA modeled after telomeres, and show that MMEJ is dependent on Polθ in human cells. Our data support a mechanism whereby Polθ facilitates end-joining and microhomology annealing then utilizes the opposing overhang as a template in trans which stabilizes the DNA synapse. Polθ exhibits a preference for DNA containing a 5’-terminal phosphate, similar to polymerases involved in non-homologous end-joining. Lastly, we identify a conserved loop domain that is essential for MMEJ and higher-order structures of Polθ which likely promote DNA synapse formation.

Roles of Residues Arg-61 and Gln-38 of Human DNA Polymerase η in Bypass of Deoxyguanosine and 7,8-Dihydro-8-oxo-2′-deoxyguanosine

“…Small angle x-ray scattering and ultracentrifugation results reveal that mammalian DNA polymerase ␤ binds with DNA in ratios of both 2:1 and 1:1 in solution (55,56). Recently, an A-family DNA polymerase (polymerase ) has been reported to be able to form a dimer, which may be important for polymerase to participate in microhomology-mediated end joining (57). Considering those examples, it is possible that the multimeric form(s) of hpol plays a specific role in certain biological processes, although further investigation will be required.…”

Polymerase θ-helicase efficiently unwinds DNA and RNA-DNA hybrids

“…Polθ has multiple documented activities in DNA replication and repair, including alt-EJ, replication repair, translesion synthesis, and replication initiation (4)(5)(6)(7)(8)10,15,31,32). Although the activities and cellular functions of Polθ-polymerase have been investigated, little is understood regarding the enzymatic activities of Polθ-helicase.…”

“…1a) (1)(2)(3). Understanding the biochemical activities and cellular functions of Polθ have become a priority because it has been found to be essential for the error-prone doublestrand break (DSB) repair pathway known as microhomology-mediated end-joining (MMEJ) or alternative end-joining (alt-EJ) (3)(4)(5)(6)(7)(8)(9). Remarkably, Polθ expression has also been shown to be important for the proliferation of cells deficient in the homologous recombination (HR) pathway, such as due to mutations in BRCA1 or BRCA2 (4,10).…”

Replication fork instability and the consequences of fork collisions from rereplication

“…MMEJ requires Pol θ, which binds to ssDNA on both ends of a DSB and aligns short 4-to 10-base-pair microhomology sequences ( Fig. 2; Chan et al 2010;Kent et al 2015;Mateos-Gomez et al 2015). Microhomologies also can be generated by Pol θ, resulting in insertions templated from sequences outside the break site (Chan et al 2010;Yu and McVey 2010;Hogg et al 2012;Kent et al 2015).…”

“…Microhomologies also can be generated by Pol θ, resulting in insertions templated from sequences outside the break site (Chan et al 2010;Yu and McVey 2010;Hogg et al 2012;Kent et al 2015). MMEJ is a highly error-prone pathway, generating deletions and insertions at the break site from microhomology alignment and extension or complex chromosome rearrangements (Chan et al 2010;Yu and McVey 2010;Hogg et al 2012;Kent et al 2015;Mateos-Gomez et al 2015;Sakofsky et al 2015).…”