title: Characterization of DNA synthesis catalyzed by bacteriophage T4 replication complexes reconstituted on synthetic circular substrates.
abstract: Replication complexes were reconstituted using the eight purified bacteriophage T4 replication proteins and synthetic circular 70-, 120- or 240-nt DNA substrates annealed to a leading-strand primer. To differentiate leading strands from lagging strands, the circular parts of the substrates lacked dCMP; thus, no dCTP was required for leading-strand synthesis and no dGTP for lagging-strand synthesis. The size of the substrates was crucial, the longer substrates supporting much more DNA synthesis. Leading and lagging strands were synthesized in a coupled manner. Specifically targeting leading-strand synthesis by decreasing the concentration of dGTP decreased the rate of extension of leading strands. However, blocking lagging-strand synthesis by lowering the dCTP concentration, by omitting dCTP altogether, by adding ddCTP, or with a single abasic site had no immediate effect on the rate of extension of leading strands.
authors.affiliation: Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709-2233, USA. kadyrov@niehs.nih.gov
authors.forename: Farid A
authors.initials: FA
authors.lastname: Kadyrov
authors.forename: John W
authors.initials: JW
authors.lastname: Drake
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology / Q000235:genetics*; D002384:Catalysis; D004261:DNA Replication; D004270:DNA, Circular / Q000096:biosynthesis*; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008957:Models, Genetic; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
chemical_list: D004270:DNA, Circular; D003845:Deoxycytosine Nucleotides; D046911:Macromolecular Substances; D014764:Viral Proteins; C024107:2'-deoxycytidine 5'-triphosphate