It is believed that replication of E. coli DNA is continuous on the leading strand and discontinuous on the lagging strand, as established in vitro experimental systems. However, there is compelling evidence that in vivo DNA replication of the leading strand is discontinuous, similar to that on the lagging strand. We are investigating the mechanism of E. coli DNA replication by isolating Okazaki fragments in agarose gels and hybridizing them to strand-specific probes. Subsequently, we will use DNA excision-repair deficient mutants to test the idea that Okazaki fragments in the leading strand are the products of DNA repair. Finally, we will try to detect RNA primers capping the 5'-ends of Okazaki fragments in the leading strand. Demonstration of a discontinuous DNA synthesis on the leading strand would provide an explanation for the uninterrupted DNA synthesis observed after DNA damage in excision repair-deficient mutants. Currently, it is believed that the replication fork is blocked by a DNA lesion in the template for the leading strand. Nevertheless, if the leading strand is made from Okazaki fragments, the replication fork should traverse the damaged area without stalling at the lesion.
| Amado, Luciana; Kuzminov, Andrei (2013) Low-molecular-weight DNA replication intermediates in Escherichia coli: mechanism of formation and strand specificity. J Mol Biol 425:4177-91 |
| Amado, Luciana; Kuzminov, Andrei (2009) Polyphosphate accumulation in Escherichia coli in response to defects in DNA metabolism. J Bacteriol 191:7410-6 |
| Amado, Luciana; Kuzminov, Andrei (2006) The replication intermediates in Escherichia coli are not the product of DNA processing or uracil excision. J Biol Chem 281:22635-46 |
