title: Replication fork stalling and checkpoint activation by a PKD1 locus mirror repeat polypurine-polypyrimidine (Pu-Py) tract.
abstract: DNA sequences prone to forming noncanonical structures (hairpins, triplexes, G-quadruplexes) cause DNA replication fork stalling, activate DNA damage responses, and represent hotspots of genomic instability associated with human disease. The 88-bp asymmetric polypurine-polypyrimidine (Pu-Py) mirror repeat tract from the human polycystic kidney disease (PKD1) intron 21 forms non-B DNA secondary structures in vitro. We show that the PKD1 mirror repeat also causes orientation-dependent fork stalling during replication in vitro and in vivo. When integrated alongside the c-myc replicator at an ectopic chromosomal site in the HeLa genome, the Pu-Py mirror repeat tract elicits a polar replication fork barrier. Increased replication protein A (RPA), Rad9, and ataxia telangiectasia- and Rad3-related (ATR) checkpoint protein binding near the mirror repeat sequence suggests that the DNA damage response is activated upon replication fork stalling. Moreover, the proximal c-myc origin of replication was not required to cause orientation-dependent checkpoint activation. Cells expressing the replication fork barrier display constitutive Chk1 phosphorylation and continued growth, i.e. checkpoint adaptation. Excision of the Pu-Py mirror repeat tract abrogates the DNA damage response. Adaptation to Chk1 phosphorylation in cells expressing the replication fork barrier may allow the accumulation of mutations that would otherwise be remediated by the DNA damage response.
authors.affiliation: Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio 45435, USA. Guoqi.Liu@yahoo.com
authors.forename: Guoqi
authors.initials: G
authors.lastname: Liu
authors.forename: Sheré
authors.initials: S
authors.lastname: Myers
authors.forename: Xiaomi
authors.initials: X
authors.lastname: Chen
authors.forename: John J
authors.initials: JJ
authors.lastname: Bissler
authors.forename: Richard R
authors.initials: RR
authors.lastname: Sinden
authors.forename: Michael
authors.initials: M
authors.lastname: Leffak
mesh_terms: D047369:Chromatin Immunoprecipitation; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004249:DNA Damage; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication; D042822:Genomic Instability; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D011687:Purines / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D050396:TRPP Cation Channels / Q000235:genetics / Q000378:metabolism*
chemical_list: D004247:DNA; D017931:DNA Primers; D016271:Proto-Oncogene Proteins c-myc; D011687:Purines; D011743:Pyrimidines; D050396:TRPP Cation Channels