ZINC-FINGER MOTIFS UNIQUE TO ARABIDOPSIS THALIANA PARALOGS RPA1C AND RPA1E ARE REQUIRED FOR RPA-DEPENDENT DNA REPAIR

Abstract RPA is a heterotrimeric ssDNA binding protein that is highly conserved across all eukaryotes. Arabidopsis ( Arabidopsis thaliana ) has five RPA1 paralogs divided into three groups (A, B, C) each with unique functions in DNA replication and repair. The group C paralogs (RPA1C and RPA1E in Arabidopsis) function specifically in DNA-damage repair and carry a C-terminal extension unique to group-C paralogs. This C-terminal extension contains a zinc finger motif (ZFM) that is highly conserved and is therefore predicted to be critical to the functionality of the paralogs during DNA damage repair. To address this, we employed a CRISPR-Cas9 strategy to specifically remove the ZFM from RPA1C or RPA1E while leaving the genes otherwise intact (termed C-ZFKO and E-ZFKO). C-ZFKO and E-ZFKO lines were challenged with DNA damaging agents, and their susceptibility was compared to both WT (Col-0) lines and to previously characterized T-DNA null mutants ( rpa1c and rpa1e ). To address the role of the respective ZFMs in homologous recombination pathways (HRR), we employed a GUS-reporter system to compare WT lines to C-ZFKO and E-ZFKO lines. We find here that C-ZFKO and E-ZFKO lines displayed hypersensitivity to DNA damaging agents at a level comparable to previously characterized T-DNA null mutants ( rpa1c and rpa1e ). When studying the rate of HRR, both C-ZFKO and rpa1c showed a drastic reduction in single-strand annealing (SSA) while E-ZFKO and rpa1e had a more modest, but still significant decrease. All mutant lines had a comparable decrease in synthesis-dependent strand annealing (SDSA) compared to WT. Thus, we show here that the respective RPA1C and RPA1E-encoded ZFM is crucial for the ability of each paralog to function during DNA damage repair.