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Nucleic Acids Research, 2002, Vol. 30, No. 20 4387-4397
© 2002 Oxford University Press

Characterization of DNA synthesis catalyzed by bacteriophage T4 replication complexes reconstituted on synthetic circular substrates

Farid A. Kadyrov* and John W. Drake

Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709-2233, USA

*To whom correspondence should be addressed. Tel: +1 919 541 3029; Fax: +1 919 541 7613; Email: kadyrov{at}niehs.nih.gov

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.


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