The PCNA interaction protein box sequence in Rad54 is an integral part of its ATPase domain and is required for efficient DNA repair and recombination

TY - JOUR

T1 - The PCNA interaction protein box sequence in Rad54 is an integral part of its ATPase domain and is required for efficient DNA repair and recombination

AU - Burgess, Rebecca C

AU - Sebesta, Marek

AU - Sisakova, Alexandra

AU - Marini, Victoria P.

AU - Lisby, Michael

AU - Damborsky, Jiri

AU - Klein, Hannah

AU - Rothstein, Rodney

AU - Krejci, Lumir

PY - 2013/12/20

Y1 - 2013/12/20

N2 - Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.

AB - Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.

KW - Adenosine Triphosphatases

KW - Amino Acid Motifs

KW - Amino Acid Sequence

KW - Chromosome Pairing

KW - Conserved Sequence

KW - DNA

KW - DNA Damage

KW - DNA Helicases

KW - DNA Mutational Analysis

KW - DNA Primers

KW - DNA Repair

KW - DNA Repair Enzymes

KW - Genomic Instability

KW - Molecular Sequence Data

KW - Mutant Proteins

KW - Mutation

KW - Proliferating Cell Nuclear Antigen

KW - Protein Binding

KW - Protein Multimerization

KW - Protein Structure, Tertiary

KW - Recombination, Genetic

KW - Saccharomyces cerevisiae

KW - Saccharomyces cerevisiae Proteins

KW - Structure-Activity Relationship

U2 - 10.1371/journal.pone.0082630

DO - 10.1371/journal.pone.0082630

M3 - Journal article

C2 - 24376557

SN - 1932-6203

VL - 8

SP - e82630

JO - PLoS Computational Biology

JF - PLoS Computational Biology

IS - 12

ER -