title: Comparison of checkpoint responses triggered by DNA polymerase inhibition versus DNA damaging agents.
abstract: To better understand the different cellular responses to replication fork pausing versus blockage, early DNA damage response markers were compared after treatment of cultured mammalian cells with agents that either inhibit DNA polymerase activity (hydroxyurea (HU) or aphidicolin) or selectively induce S-phase DNA damage responses (the DNA alkylating agents, methyl methanesulfonate (MMS) and adozelesin). These agents were compared for their relative abilities to induce phosphorylation of Chk1, H2AX, and replication protein A (RPA), and intra-nuclear focalization of gamma-H2AX and RPA. Treatment by aphidicolin and HU resulted in phosphorylation of Chk1, while HU, but not aphidicolin, induced focalization of gamma-H2AX and RPA. Surprisingly, pre-treatment with aphidicolin to stop replication fork progression, did not abrogate HU-induced gamma-H2AX and RPA focalization. This suggests that HU may act on the replication fork machinery directly, such that fork progression is not required to trigger these responses. The DNA-damaging fork-blocking agents, adozelesin and MMS, both induced phosphorylation and focalization of H2AX and RPA. Unlike adozelesin and HU, the pattern of MMS-induced RPA focalization did not match the BUdR incorporation pattern and was not blocked by aphidicolin, suggesting that MMS-induced damage is not replication fork-dependent. In support of this, MMS was the only reagent used that did not induce phosphorylation of Chk1. These results indicate that induction of DNA damage checkpoint responses due to adozelesin is both replication fork and fork progression dependent, induction by HU is replication fork dependent but progression independent, while induction by MMS is independent of both replication forks and fork progression.
authors.affiliation: Department of Microbiology, Witebsky Center for Microbial Pathogenesis & Immunology, School of Medicine & Biomedical Sciences, 138 Farber Hall, SUNY at Buffalo, Buffalo, NY 14214-3000, USA.
authors.forename: Jen-Sing
authors.initials: JS
authors.lastname: Liu
authors.forename: Shu-Ru
authors.initials: SR
authors.lastname: Kuo
authors.forename: Thomas
authors.initials: T
authors.lastname: Melendy
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D016590:Aphidicolin / Q000494:pharmacology; D064007:Ataxia Telangiectasia Mutated Proteins; D001572:Benzofurans; D018797:Cell Cycle Proteins; D000071877:Checkpoint Kinase 1; D020552:Comet Assay; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology; D053138:Cyclohexenes; D004249:DNA Damage / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000080890:Duocarmycins; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051716:Replication Protein A; D016196:S Phase / Q000187:drug effects*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins
chemical_list: D018906:Antineoplastic Agents, Alkylating; D001572:Benzofurans; D018797:Cell Cycle Proteins; D003509:Cyclohexanecarboxylic Acids; D053138:Cyclohexenes; D004268:DNA-Binding Proteins; D000080890:Duocarmycins; D004791:Enzyme Inhibitors; C468783:H2AX protein, human; D006657:Histones; D007211:Indoles; D019384:Nucleic Acid Synthesis Inhibitors; C497167:RPA1 protein, human; D051716:Replication Protein A; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D016590:Aphidicolin; C060090:adozelesin; D008741:Methyl Methanesulfonate; D011494:Protein Kinases; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D006918:Hydroxyurea