Gene Targeting in Plants: Roles of the ATM and ATR protein kinases

"Gene Targeting" is a highly sophisticated technique for functional genomic analysis and for highly accurate genetic modification by which pre-selected genes within a complex genome can be altered through targeted deletion ("knock-out"), introduction of reporter fusions ("knock-in") or single nucleotide modification "point mutations".

Among model plants, the moss Physcomitrella patens is unique in its ability to efficiently integrate transforming DNA preferentially at homologous loci by homologous recombination (“Gene Targeting”). Wider exploitation of Gene Targeting requires a good understanding of the molecular mechanisms by which this occurs. The working hypothesis is that transforming DNA is recruited by endogenous mechanisms for the repair of DNA-double-strand breaks and that in P. patens, the predominating pathway is an homology-dependent (HR) pathway, rather than the non-homologous end-joining (NHEJ) pathway favoured by most complex eukaryotes.

The selection of DSB-repair pathways is regulated by the protein kinases, “ATM” and “ATR”. Upon perception of DSBs, these initiate a phosphorylation cascade invoking a cell-cycle checkpoint required for completing DNA repair before cell division occurs. In mammalian cells, ATM is primarily associated with a G1/S checkpoint and ATR with a G2/M checkpoint, acting through the checkpoint kinases CHK2 and CHK1, respectively. Cell cycle arrest at G2/M is associated with HR dependent repair, whilst arrest at G1/S is associated with NHEJ. In plants, no obvious homologues of CHK1 and CHK2 have been identified.

This project will identify the targets of ATM and ATR phosphorylation following DNA damage and during transformation in wild-type and mutant strains of P.patens, using proteomic technology, and analyse the functions of these targets in vivo by homologous recombination-mediated mutagenesis.

Objectives:
(i) To identify proteins phosphorylated in P. patens following DNA damage and transgene delivery.
(ii) To identify ATM and ATR targets by comparative analysis proetin phosphorylation in gene-targeted atm and atr mutants.
(iii) To functionally test the roles of candidate proteins identified in (i) and (ii) by targeted mutagenesis.

Novelty:
State-of-the art methodology for phosphoproteomic profiling will be combined with sophisticated techniques for genetic manipulation in a highly tractable model organism.

Timeliness:
Extensive genetic and genomic resources have been developed for P. patens including a fully sequenced genome and tools for forward and reverse genetics. The host laboratory is supported by BBSRC funding to characterise the mechanism of plant gene targeting.