Molecular hand-off mechanisms during lagging strand replication

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DNA replication is the process of copying one DNA molecule to form two identical ones. It is highly conserved at the mechanistic level across evolution. It comprises a highly complex set of biochemical reactions carried out by intricate enzyme assemblies, coordinated within the cell cycle and in response to external and internal cellular signals. Insights into the replication processes at the molecular level will provide opportunities to modulate and intervene in replication; rapidly dividing cells need to replicate their DNA prior to dividing, and targeting components of the replication process is potentially a very powerful strategy in the treatment of cancer and microbial infections. Targeting DNA replication of pathogenic bacteria and viruses is a clinical reality but it is a grossly underexplored area of drug development. DNA replication is fundamental to a huge range of molecular biological and biochemical applications, and provides many potential targets for rational drug design in the treatment pathogenic infections. Without understanding the chemistry of DNA replication we will not be able to explore new drug targets.A large group of pathogenic and non-pathogenic bacteria use two different enzymes (DNA polymerases) to copy the parental DNA to form nascent DNA. These two enzymes are known as DnaE and PolC. DnaE is a relatively poor enzyme prone to making mistakes, while PolC is a powerful enzyme with extremely high fidelity. The polymerases cannot synthesize new DNA using its building blocks, known as deoxynucleotide tri-phosphates (dNTPs for short). Instead, the parental DNA is first copied in a short stretch of an alternative form of a nucleic acid, known as RNA, which is synthesized from its building blocks ribonucleotide tri-phosphates (NTPs for short) by another enzyme known as primase. The short RNA stretch (fragment) is then extended by DnaE to form an RNA-DNA hybrid fragment which is then handed off to the powerful and accurate PolC to be extended further by copying the parental template strand. This process, therefore, involves two molecular hand-off mechanisms; First the primase synthesizes the short RNA and hands it off to DnaE for initial extension and second the DnaE forms the RNA-DNA hybrid nucleic acid and hands it off to PolC. Even with all our relatively detailed knowledge of DNA replication we still know nothing about the molecular details of these two hand-off mechanisms. Here, we aim to study these mechanisms and reveal their molecular details. In order to do this we have purified large quantities of these proteins and set up a novel coupled assay. With this assay we can detect simultaneously, unwinding of the parental double stranded DNA template by the enzyme helicase (DnaC), synthesis of the RNA primer by the enzyme primase (DnaG) and initial extension of the RNA primer to form the RNA-DNA hybrid by the enzyme polymerase (DnaE).We have established that these three proteins interact with each other to form a functional complex. The activities of all three proteins are coordinated within this complex. We also have evidence suggesting that PolC corrects the mistakes made by DnaE in trans. Using this powerful minimal coupled assay we will now study the molecular details of the DnaG-DnaE and DnaE-PolC hand off mechanisms. We will also build structural models of the interacting proteins to gain unprecedented detailed understanding of the structural principles that underpin these two hand-off mechanisms.