The Importance of ATM and ATR in Physcomitrella patens DNA Damage Repair, Development, and Gene Targeting.

Martin Martens,Edelgard Wendeler,Bernd Reiss,Ralf Horres

doi:10.3390/genes11070752

Abstract

Coordinated by ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR), two highly conserved kinases, DNA damage repair ensures genome integrity and survival in all organisms. The Arabidopsis thaliana (A. thaliana) orthologues are well characterized and exhibit typical mammalian characteristics. We mutated the Physcomitrella patens (P. patens) PpATM and PpATR genes by deleting functionally important domains using gene targeting. Both mutants showed growth abnormalities, indicating that these genes, particularly PpATR, are important for normal vegetative development. ATR was also required for repair of both direct and replication-coupled double-strand breaks (DSBs) and dominated the transcriptional response to direct DSBs, whereas ATM was far less important, as shown by assays assessing resistance to DSB induction and SuperSAGE-based transcriptomics focused on DNA damage repair genes. These characteristics differed significantly from the A. thaliana genes but resembled those in yeast (Saccharomyces cerevisiae). PpATR was not important for gene targeting, pointing to differences in the regulation of gene targeting and direct DSB repair. Our analysis suggests that ATM and ATR functions can be substantially diverged between plants. The differences in ATM and ATR reflect the differences in DSB repair pathway choices between A. thaliana and P. patens, suggesting that they represent adaptations to different demands for the maintenance of genome stability.

Highlights

DNA damage caused by intrinsic and extrinsic factors constantly challenges genome stability [1].Repair of DNA damage employs a complex network of interacting pathways, the DNA damage response (DDR), comprising DNA damage sensing, signal integration, and signal transduction processes that activate diverse DNA damage repair pathways and their connections to DNA replication and cell-cycle control [2,3,4,5]
double-strand breaks (DSBs) repair occurs by two principal pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR) [6,7], processes that differ in their requirement for a homologous template, their precision and speed of repair, and their dependence on the state of the cell-cycle [5,8,9]
The identification of ATM and ATR is described in detail in the Materials and Methods section and it was essentially confirmed that the gene models in the latest P. patens chromosome-scale assembly, Pp3c2_23700V3.1 (PpATM) and Pp3c6_3460V3.1 (PpATR) [33], are the correct P. patens orthologues

Summary

Introduction

DNA damage caused by intrinsic and extrinsic factors constantly challenges genome stability [1].Repair of DNA damage employs a complex network of interacting pathways, the DNA damage response (DDR), comprising DNA damage sensing, signal integration, and signal transduction processes that activate diverse DNA damage repair pathways and their connections to DNA replication and cell-cycle control [2,3,4,5]. Arabidopsis thaliana atm mutants are highly sensitive to IR and methyl methanesulfonate (MMS), but not UV-B [13] and the radiomimetic bleomycin [14], indicating the importance of ATM in the repair of direct DSBs. Atatr mutants are less sensitive to IR [15] but are highly sensitive to agents inducing replication-coupled DSBs such as replication blocking agents (aphidicolin, UV-B, hydroxyurea) [15,16] and the inter-strand crosslinking agent mitomycin C [14]. Atatr mutants are less sensitive to IR [15] but are highly sensitive to agents inducing replication-coupled DSBs such as replication blocking agents (aphidicolin, UV-B, hydroxyurea) [15,16] and the inter-strand crosslinking agent mitomycin C [14] These resistance patterns suggest a comparable specialization of ATM and ATR functions in plants [17]. ATM plays a leading role in the control of the transcriptional response to direct DSBs in A. thaliana, whereas ATR is of minor importance [15,18]

Methods

Results

Discussion

Conclusion