title: Translocation and stability of replicative DNA helicases upon encountering DNA-protein cross-links.
abstract: DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0-14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15-36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Nakano
authors.forename: Mayumi
authors.initials: M
authors.lastname: Miyamoto-Matsubara
authors.forename: Mahmoud I
authors.initials: MI
authors.lastname: Shoulkamy
authors.forename: Amir M H
authors.initials: AM
authors.lastname: Salem
authors.forename: Seung Pil
authors.initials: SP
authors.lastname: Pack
authors.forename: Yukio
authors.initials: Y
authors.lastname: Ishimi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ide
mesh_terms: D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D004265:DNA Helicases / Q000737:chemistry* / Q000502:physiology*; D053488:DnaB Helicases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D026161:MADS Domain Proteins / Q000378:metabolism; D008957:Models, Genetic; D009473:Neuronal Plasticity; D009841:Oligonucleotides / Q000235:genetics; D011485:Protein Binding; D021381:Protein Transport; D013569:Synapses / Q000378:metabolism; D013997:Time Factors; D014981:Xenopus
chemical_list: D003432:Cross-Linking Reagents; D026161:MADS Domain Proteins; D009841:Oligonucleotides; D004247:DNA; C506730:dnaB protein, E coli; D004265:DNA Helicases; D053488:DnaB Helicases