Homologous recombination and nonhomologous end-joining repair pathways regulate fragile site stability.
Common fragile sites are specific loci that form gaps and constrictions on metaphase chromosomes exposed to replication stress, which slows DNA replication. These sites have a role in chromosomal rearrangements in tumors; however, the molecular mechanism of their expression is unclear. Here we show that replication stress leads to focus formation of Rad51 and phosphorylated DNA-PKcs, key components of the homologous recombination (HR) and nonhomologous end-joining (NHEJ), double-strand break (DSB) repair pathways, respectively. Down-regulation of Rad51, DNA-PKcs, or Ligase IV, an additional component of the NHEJ repair pathway, leads to a significant increase in fragile site expression under replication stress. Replication stress also results in focus formation of the DSB markers, MDC1 and gammaH2AX. These foci colocalized with those of Rad51 and phospho-DNA-PKcs. Furthermore, gammaH2AX and phospho-DNA-PKcs foci were localized at expressed fragile sites on metaphase chromosomes. These findings suggest that DSBs are formed at common fragile sites as a result of replication perturbation. The repair of these breaks by both HR and NHEJ pathways is essential for chromosomal stability at these sites.
Schwartz
Michal
M
Department of Genetics, The Life Sciences Institute, The Hebrew University, Jerusalem, Israel.
Zlotorynski
Eitan
E
Goldberg
Michal
M
Ozeri
Efrat
E
Rahat
Ayelet
A
le Sage
Carlos
C
Chen
Benjamin P C
BP
Chen
David J
DJ
Agami
Reuven
R
Kerem
Batsheva
B
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D043283:Chromosome Fragile Sites / Q000235:genetics* / Q000502:physiology; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000502:physiology; D004260:DNA Repair / Q000502:physiology*; D051747:DNA-Activated Protein Kinase / Q000502:physiology; D004268:DNA-Binding Proteins / Q000502:physiology; D006367:HeLa Cells; D006657:Histones / Q000502:physiology; D006801:Humans; D008677:Metaphase / Q000502:physiology; D009687:Nuclear Proteins / Q000502:physiology; D010766:Phosphorylation; D051135:Rad51 Recombinase / Q000502:physiology; D011995:Recombination, Genetic / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics*; D015534:Trans-Activators / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; C413163:MDC1 protein, human; D009687:Nuclear Proteins; D015534:Trans-Activators; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D051135:Rad51 Recombinase; D011088:DNA Ligases; D000072481:DNA Ligase ATP