Inhibition of Replication Fork Formation and Progression: Targeting the Replication Initiation and Primosomal Proteins

Abstract

Over 1.2 million deaths are attributed to multi-drug-resistant (MDR) bacteria each year. Persistence of MDR bacteria is primarily due to the molecular mechanisms that permit fast replication and rapid evolution. As many pathogens continue to build resistance genes, current antibiotic treatments are being rendered useless and the pool of reliable treatments for many MDR-associated diseases is thus shrinking at an alarming rate. In the development of novel antibiotics, DNA replication is still a largely underexplored target. This review summarises critical literature and synthesises our current understanding of DNA replication initiation in bacteria with a particular focus on the utility and applicability of essential initiation proteins as emerging drug targets. A critical evaluation of the specific methods available to examine and screen the most promising replication initiation proteins is provided.

Keywords: DNA replication; DnaA; antibiotics; bacteria; helicase; high-throughput screening; primase; replication fork; replisome.

Figure 1

Replication fork progression from initiation to termination. (A) Multi-fork replication in fast growing bacteria. Multiple replisomes may load sequentially at the origin site (initiation at oriC in E. coli), as a result of reduced generation time. (B) Bacterial replisome during fork progression (elongation stage). As the helicase unwinds the replication bubble, RNA primers are synthesised for extension by DNA Pol III*. Polymerase activity is mediated by the β sliding clamp and its clamp loading complex (CLC). Exposed single-stranded DNA (ssDNA depicted here using a single line) is protected by single-stranded binding protein (SSB) throughout this process. In E. coli, adjacent Okazaki fragments on the lagging strand are joined by DNA Pol I and DNA Ligase A (not depicted). Topoisomerases release tension in the double helix as the fork progresses. Type I topoisomerases achieve this by nicking one strand within the double helix, whereas type II topoisomerases disentangle double-stranded DNA (dsDNA) by cutting both strands and passing one duplex DNA through the other to remove loops created by supercoiling. Examples of inhibitors indicated by numbered boxes (1–5) are described in Table 2.

Figure 2

Initiation of DNA replication and the formation of the primosome at the origin (oriC in E. coli). (A) The initiator protein (DnaA) instigates chromosome replication through its binding to specific motifs within the DnaA-oligomerisation region (DOR), melting the DNA at the DNA unwinding element (DUE) to form the open complex. (B) Two hexameric helicases (DnaB₆) as well as SSB tetramers (not depicted) are recruited to expand the replication bubble. (C) Final stage of initiation involving recruitment of the primase (DnaG) and initial RNA primer synthesis for the leading strand.