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Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression
Half of human genome is made 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 (BACs) in Xenopus laevis egg extr...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939857/ https://www.ncbi.nlm.nih.gov/pubmed/27111843 http://dx.doi.org/10.1038/ncb3344 |
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author | Aze, Antoine Sannino, Vincenzo Soffientini, Paolo Bachi, Angela Costanzo, Vincenzo |
author_facet | Aze, Antoine Sannino, Vincenzo Soffientini, Paolo Bachi, Angela Costanzo, Vincenzo |
author_sort | Aze, Antoine |
collection | PubMed |
description | Half of human genome is made 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 (BACs) in Xenopus laevis egg extract. Using this approach we characterized 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 among which 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 inability of 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 on our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions. |
format | Online Article Text |
id | pubmed-4939857 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
record_format | MEDLINE/PubMed |
spelling | pubmed-49398572016-10-25 Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression Aze, Antoine Sannino, Vincenzo Soffientini, Paolo Bachi, Angela Costanzo, Vincenzo Nat Cell Biol Article Half of human genome is made 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 (BACs) in Xenopus laevis egg extract. Using this approach we characterized 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 among which 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 inability of 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 on our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions. 2016-04-25 2016-06 /pmc/articles/PMC4939857/ /pubmed/27111843 http://dx.doi.org/10.1038/ncb3344 Text en Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms |
spellingShingle | Article Aze, Antoine Sannino, Vincenzo Soffientini, Paolo Bachi, Angela Costanzo, Vincenzo Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression |
title | Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression |
title_full | Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression |
title_fullStr | Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression |
title_full_unstemmed | Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression |
title_short | Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression |
title_sort | centromeric dna replication reconstitution reveals dna loops and atr checkpoint suppression |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939857/ https://www.ncbi.nlm.nih.gov/pubmed/27111843 http://dx.doi.org/10.1038/ncb3344 |
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