Mechanisms of bacterial DNA replication restart

Abstract

Multi-protein DNA replication complexes called replisomes perform the essential process of copying cellular genetic information prior to cell division. Under ideal conditions, replisomes dissociate only after the entire genome has been duplicated. However, DNA replication rarely occurs without interruptions that can dislodge replisomes from DNA. Such events produce incompletely replicated chromosomes that, if left unrepaired, prevent the segregation of full genomes to daughter cells. To mitigate this threat, cells have evolved 'DNA replication restart' pathways that have been best defined in bacteria. Replication restart requires recognition and remodeling of abandoned replication forks by DNA replication restart proteins followed by reloading of the replicative DNA helicase, which subsequently directs assembly of the remaining replisome subunits. This review summarizes our current understanding of the mechanisms underlying replication restart and the proteins that drive the process in Escherichia coli (PriA, PriB, PriC and DnaT).

Figure 1.

DNA replication and forked DNA structures that can be recognized by the replication restart machinery. (A) Cartoon representation of DNA replication in E. coli from the origin of replication (oriC) to the terminus (ter), depicting replication fork arrest and collapse that often occurs during this process. (B) Schematic of the DNA fork structures present at abandoned replication forks and other fork types that replication restart can recognize. These additional fork types are found during DNA repair processes, certain phage and plasmid DNA replication initiation, and during/after transcription. DNA is indicated in black (parental) and grey (nascent) with RNA shown in red. SSB is shown in orange.

Figure 2.

Structures of the bacterial DNA replication restart proteins. (A) Domain architecture and crystal structure of Klebsiella pneumoniae PriA (pdb: 4NL4; ADP in red sticks and Zn²⁺ in gray spheres (43)), with each structural domain individually colored, overlayed with the structure of the E. coli 3′ BD cocrystalized with dinucleotide (pale red and black respectively; pdb: 2D7G, (80)). (B) Structure of the E. coli PriB dimer (pdb: 1WOC, (183)) shown in magenta, overlayed with the structure of E. coli PriB dimer cocrystalized with ssDNA (grey and black respectively; pdb: 2CCZ, (119)). Inset: two DNA-binding domains of E. coli SSB bound to ssDNA (grey and black respectively, pdb: 1EYG, (184)). (C) Left: The solution NMR structure of the 98 N-terminal residues of E. coli PriC shown in wheat (pdb: 2RT6, (129)). Right: The solution NMR structure of full-length Cronobacter sakazakii PriC protein shown in brown (pdb: 2NCJ,(127)). (D) The solution NMR structure of the C-terminal 90 residues of the E. coli DnaT monomer shown in dark blue (middle; pdb: 2RU8, (96)) overlayed with the crystal structure of E. coli DnaT 84–153 bound to dT10 (the five subunits in the oligomer shown in cyan, where the middle one has been overlayed with 2RU8 and the outer two are shown with transparent grey surface, and DNA in black; pdb: 4OU7, (98)). Domain architecture shown to left of each structure, where grey sections indicated the protein sections not included and/or resolved in the structure(s) at right.

Figure 3.

DNA replication restart pathways in Escherichia coli. E. coli and related bacteria possess three functional pathways for DNA replication restart, with the fourth column likely only occurring in vitro. All pathways serve to reload the replicative helicase DnaB on sites far removed from the origin of replication in a DNA structure-dependent manner. Key steps in the process are separated by row. Abandoned replication forks (various forms depicted in top row, depending on whether the strands are dsDNA or SSB-coated ssDNA gaps) are recognized by either by PriA or PriC (second row; SSB-interaction/remodeling not shown). Remodeling of the fork through helicase activity (third row) or SSB-interaction may allow for or/and proceed subsequent protein-protein interactions (fourth row). Replication restart ends with DnaB loading (bottom). Note that for simplicity PriA and PriC recognition of a select/preferred fork type is shown. PriA helicase activity is not required on many fork types and two PriA molecules could be shown on a fork. Rep helicase may function before PriC recognition on the first/left fork type. Helicase activity likely does not function exactly in this order and could occur during all steps.