POLQ plays a key role in the repair of CRISPR/Cas9‐induced double‐stranded breaks in the moss Physcomitrella patens

Abstract

Summary Double‐stranded breaks can be repaired by different mechanisms such as homologous recombination (HR), classical nonhomologous end joining (C‐NHEJ) and alternative end joining (Alt‐EJ). Polymerase Q (POLQ) has been proposed to be the main factor involved in Alt‐EJ‐mediated DNA repair. Here we describe the role of POLQ in DNA repair and gene targeting in Physcomitrella patens. The disruption of the POLQ gene does not influence the genetic stability of P. patens nor its development. The polq mutant shows the same sensitivity as wild‐type towards most of the genotoxic agents tested (ultraviolet (UV), methyl methanesulfonate (MMS) and cisplatin) with the notable exception of bleomycin for which it shows less sensitivity than the wild‐type. Furthermore, we show that POLQ is involved in the repair of CRISPR‐Cas9‐induced double‐stranded breaks in P. patens. We also demonstrate that POLQ is a potential competitor and/or inhibitor of the HR repair pathway. This finding has a consequence in terms of genetic engineering, as in the absence of POLQ the frequency of gene targeting is significantly increased and the number of clean two‐sided HR‐mediated insertions is enhanced. Therefore, the control of POLQ activity in plants could be a useful strategy to optimize the tools of genome engineering for plant breeding.

The plant DNA polymerase theta is essential for the repair of replication‐associated DNA damage

“…In plants, the situation seems less clear, as Arabidopsis teb mutants grown under our standard laboratory conditions display very variable phenotypic defects and rice mutants grow and develop normally (Nishizawa‐Yokoi et al ., 2020). Likewise, in the moss Physcomitrium patens , loss of Pol θ does not affect development or genetic stability (Mara et al ., 2019). The latter observation may relate to the fact that the most prominent DNA repair pathway in moss cells is HR rather than Alt‐NHEJ: in P. patens , mutants deficient in the RAD51 protein, which is required for HR, show developmental defects and hypersensitivity to DNA‐damaging agents (Markmann‐Mulisch et al ., 2007).…”

The XPF-ERCC1 Complex Is Essential for Genome Stability and Is Involved in the Mechanism of Gene Targeting in Physcomitrella patens

“…One hypothesis to explain the role of the complex in the formation of TGI and concatemers events could be its potential role in the handling of the looped-out heteroduplex intermediates (Supplementary Figure S6) formed during the invasion process in presence of the concatemers that are produced before integration at the targeted APT locus. In this context, and taking into consideration our recent data showing the involvement of the POLQ protein in TGI events formation in P. patens (Mara et al, 2019), it would be interesting to check for a potential interaction between the XPF-ERCC1 complex and the Alt-EJ pathway for the targeted integration of ends-out construct in this moss. Such a cross-talk between the RAD1-RAD10 complex and the Alt-EJ repair pathway, that, like the SSA pathway, involves the removal of non-homologous 3′ tails, has already been proposed in yeast and more recently in animals (Ma et al, 2003; Sallmyr and Tomkinson, 2018).…”

Mechanisms of Genome Maintenance in Plants: Playing It Safe With Breaks and Bumps

“…Overall, these studies point toward the existence of a different mechanism for the repair of DSBs in plants. However, in mosses, the repair of DSBs predominantly occurs through HRR ( Mara et al, 2019 ). Pol q deletion mutants do not show any developmental or genetic instability phenotype in mosses.…”