Break-induced ATR and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent nucleotide synthesis promotes homologous recombination repair in fission yeast.
Nucleotide synthesis is a universal response to DNA damage, but how this response facilitates DNA repair and cell survival is unclear. Here we establish a role for DNA damage-induced nucleotide synthesis in homologous recombination (HR) repair in fission yeast. Using a genetic screen, we found the Ddb1-Cul4(Cdt)² ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1-Cul4(Cdt)² ubiquitin ligase complex is required for degradation of Spd1, an inhibitor of RNR in fission yeast. Accordingly, deleting spd1(+) suppressed the DNA damage sensitivity and the reduced HR efficiency associated with loss of ddb1(+) or cdt2(+). Furthermore, we demonstrate a role for nucleotide synthesis in postsynaptic gap filling of resected ssDNA ends during HR repair. Finally, we define a role for Rad3 (ATR) in nucleotide synthesis and HR through increasing Cdt2 nuclear levels in response to DNA damage. Our findings support a model in which break-induced Rad3 and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent Spd1 degradation and RNR activation promotes postsynaptic ssDNA gap filling during HR repair.
Department of Oncology, Cancer Research UK-Medical Research Council Gray Institute for Radiation Oncology and Biology, University of Oxford, United Kingdom.
Jennifer
J
Moss
Helen
H
Tinline-Purvis
Carol A
CA
Walker
Lisa K
LK
Folkes
Michael R
MR
Stratford
Jacqueline
J
Hayles
Kwang-Lae
KL
Hoe
Dong-Uk
DU
Kim
Han-Oh
HO
Park
Stephen E
SE
Kearsey
Oliver
O
Fleck
Christian
C
Holmberg
Olaf
O
Nielsen
Timothy C
TC
Humphrey
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017353:Gene Deletion; D009711:Nucleotides / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011995:Recombination, Genetic; D012264:Ribonucleotide Reductases / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C506543:Cdt2 protein, S pombe; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C500125:Ddb1 protein, S pombe; D009711:Nucleotides; D029702:Schizosaccharomyces pombe Proteins; C102445:Spd1 protein, S pombe; D012264:Ribonucleotide Reductases; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C456840:rad3 protein, S pombe