Article

Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Molecular Cell (Impact Factor: 15.28). 06/2003; 11(5):1349-60.
Source: PubMed

ABSTRACT DNA primases are template-dependent RNA polymerases that synthesize oligoribonucleotide primers that can be extended by DNA polymerase. The bacterial primases consist of zinc binding and RNA polymerase domains that polymerize ribonucleotides at templating sequences of single-stranded DNA. We report a crystal structure of bacteriophage T7 primase that reveals its two domains and the presence of two Mg(2+) ions bound to the active site. NMR and biochemical data show that the two domains remain separated until the primase binds to DNA and nucleotide. The zinc binding domain alone can stimulate primer extension by T7 DNA polymerase. These findings suggest that the zinc binding domain couples primer synthesis with primer utilization by securing the DNA template in the primase active site and then delivering the primed DNA template to DNA polymerase. The modular architecture of the primase and a similar mechanism of priming DNA synthesis are likely to apply broadly to prokaryotic primases.

0 Bookmarks
 · 
58 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Replication of DNA plays a central role in transmitting hereditary information from cell to cell. To achieve reliable DNA replication, multiple proteins form a stable complex, known as the replisome, enabling them to act together in a highly coordinated fashion. Over the past decade, the roles of the various proteins within the replisome have been determined. Although many of their interactions have been characterized, it remains poorly understood how replication proteins enter and leave the replisome. In this study, we visualize fluorescently labeled bacteriophage T7 DNA polymerases within the replisome while we simultaneously observe the kinetics of the replication process. This combination of observables allows us to monitor both the activity and dynamics of individual polymerases during coordinated leading- and lagging-strand synthesis. Our data suggest that lagging-strand polymerases are exchanged at a frequency similar to that of Okazaki fragment synthesis and that two or more polymerases are present in the replisome during DNA replication. Our studies imply a highly dynamic picture of the replisome with lagging-strand DNA polymerases residing at the fork for the synthesis of only a few Okazaki fragments. Further, new lagging-strand polymerases are readily recruited from a pool of polymerases that are proximally bound to the replisome and continuously replenished from solution.
    Proceedings of the National Academy of Sciences 03/2014; · 9.81 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A cell can be thought of as a highly sophisticated micro factory: in a pool of billions of molecules - metabolites, structural proteins, enzymes, oligonucleotides - multi-subunit complexes assemble to perform a large number of basic cellular tasks, such as DNA replication, RNA/protein synthesis or intracellular transport. By purifying single components and using them to reconstitute molecular processes in a test tube, researchers have gathered crucial knowledge about mechanistic, dynamic and structural properties of biochemical pathways. However, to sort this information into an accurate cellular road map, we need to understand reactions in their relevant context within the cellular hierarchy, which is at the individual molecule level within a crowded, cellular environment. Reactions occur in a stochastic fashion, have short-lived and not necessarily well-defined intermediates, and dynamically form functional entities. With the use of single-molecule techniques these steps can be followed and detailed kinetic information that otherwise would be hidden in ensemble averaging can be obtained. One of the first complex cellular tasks that have been studied at the single-molecule level is the replication of DNA. The replisome, the multi-protein machinery responsible for copying DNA, is built from a large number of proteins that function together in an intricate and efficient fashion allowing the complex to tolerate DNA damage, roadblocks or fluctuations in subunit concentration. In this review, we summarize advances in single-molecule studies, both in vitro and in vivo, that have contributed to our current knowledge of the mechanistic principles underlying DNA replication.
    Chemical Society Reviews 01/2014; · 24.89 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Replisomes are multi-protein complexes that coordinate the synthesis of leading and lagging DNA strands to increase replication efficiency and reduce DNA strand breaks caused by stalling of replication forks. The bacteriophage T7 replisome is an economical machine that requires only four proteins for processive, coupled synthesis of two DNA strands. Here we characterize a complex between T7 primase-helicase and DNA polymerase on DNA that was trapped during the initiation of Okazaki fragment synthesis from an RNA primer. This priming complex consists of two DNA polymerases and a primase-helicase hexamer that assemble on the DNA template in an RNA-dependent manner. The zinc binding domain of the primase-helicase is essential for trapping the RNA primer in complex with the polymerase, and a unique loop located on the thumb of the polymerase also stabilizes this primer extension complex. Whereas one of the polymerases engages the primase-helicase and RNA primer on the lagging strand of a model replication fork, the second polymerase in the complex is also functional and can bind a primed template DNA. These results indicate that the T7 primase-helicase specifically engages two copies of DNA polymerase, which would enable the coordination of leading and lagging strand synthesis at a replication fork. Assembly of the T7 replisome is driven by intimate interactions between the DNA polymerase and multiple subunits of the primase-helicase hexamer.
    Biochemistry 05/2013; · 3.38 Impact Factor

Full-text

View
0 Downloads
Available from