Abstract
Replication and repair of genomic DNA requires the actions of multiple enzymatic functions that must be coordinated in order to ensure efficient and accurate product formation. Here, we have used single-molecule FRET microscopy to investigate the physical basis of functional coordination in DNA polymerase I (Pol I) from Escherichia coli, a key enzyme involved in lagging-strand replication and base excision repair. Pol I contains active sites for template-directed DNA polymerization and 5' flap processing in separate domains. We show that a DNA substrate can spontaneously transfer between polymerase and 5' nuclease domains during a single encounter with Pol I. Additionally, we show that the flexibly tethered 5' nuclease domain adopts different positions within Pol I-DNA complexes, depending on the nature of the DNA substrate. Our results reveal the structural dynamics that underlie functional coordination in Pol I and are likely relevant to other multi-functional DNA polymerases.
Highlights
DNA polymerases from many organisms need to coordinate multiple enzymatic activities to achieve accurate and efficient replication and repair of DNA, while avoiding the formation of mutagenic or unstable DNA intermediates (Reha-Krantz, 2010; Bebenek and Ziuzia-Graczyk, 2018)
Individual encounters between polymerase I (Pol I), present in solution, and the surface-immobilized DNA substrates were visualized by single-molecule Forster resonance energy transfer (smFRET) microscopy
The 5’ nuc domain of Pol I fulfils a key function during DNA replication and repair, cleaving 5’ flaps that arise from strand displacement synthesis (Figure 1A)
Summary
DNA polymerases from many organisms need to coordinate multiple enzymatic activities to achieve accurate and efficient replication and repair of DNA, while avoiding the formation of mutagenic or unstable DNA intermediates (Reha-Krantz, 2010; Bebenek and Ziuzia-Graczyk, 2018). Pol I plays an important role in lagging strand DNA replication in E. coli (Balakrishnan and Bambara, 2013; Okazaki et al, 1971). During this complex process, short RNA primers anneal to the lagging strand and are extended by DNA primase, producing fused RNA-DNA strands (Okazaki fragments). The nascent DNA portion is subsequently extended by Pol I, until the growing strand encounters another Okazaki fragment lying downstream, displacing the 5’ end and forming an RNA flap. The same processing steps are performed by Pol I during DNA base excision repair, in which case the displaced strand is composed of DNA (Imai et al, 2007)
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