Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.
Half of the human genome is made up of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using bacterial artificial chromosomes in Xenopus laevis egg extract. Using this approach we characterized the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication-dependent enrichment of a network of DNA repair factors including the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR-dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to the inability of the single-stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of topoisomerase I-dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.
DNA Metabolism Laboratory, IFOM, The FIRC Institute of Molecular Oncology, Via Adamello 16, Milan 20139, Italy.
Antoine
A
Aze
DNA Metabolism Laboratory, IFOM, The FIRC Institute of Molecular Oncology, Via Adamello 16, Milan 20139, Italy.
Vincenzo
V
Sannino
DNA Metabolism Laboratory, IFOM, The FIRC Institute of Molecular Oncology, Via Adamello 16, Milan 20139, Italy.
Paolo
P
Soffientini
DNA Metabolism Laboratory, IFOM, The FIRC Institute of Molecular Oncology, Via Adamello 16, Milan 20139, Italy.
Angela
0000-0003-4842-6556
A
Bachi
DNA Metabolism Laboratory, IFOM, The FIRC Institute of Molecular Oncology, Via Adamello 16, Milan 20139, Italy.
Vincenzo
V
Costanzo
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism*; D059447:Cell Cycle Checkpoints / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002503:Centromere / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D011494:Protein Kinases / Q000378:metabolism; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D029867:Xenopus Proteins; D004247:DNA; D011494:Protein Kinases; D064007:Ataxia Telangiectasia Mutated Proteins