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. 2013 Nov 28;503(7477):544-547.
doi: 10.1038/nature12650. Epub 2013 Nov 3.

Accelerated Growth in the Absence of DNA Replication Origins

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Free PMC article

Accelerated Growth in the Absence of DNA Replication Origins

Michelle Hawkins et al. Nature. .
Free PMC article

Abstract

DNA replication initiates at defined sites called origins, which serve as binding sites for initiator proteins that recruit the replicative machinery. Origins differ in number and structure across the three domains of life and their properties determine the dynamics of chromosome replication. Bacteria and some archaea replicate from single origins, whereas most archaea and all eukaryotes replicate using multiple origins. Initiation mechanisms that rely on homologous recombination operate in some viruses. Here we show that such mechanisms also operate in archaea. We use deep sequencing to study replication in Haloferax volcanii and identify four chromosomal origins of differing activity. Deletion of individual origins results in perturbed replication dynamics and reduced growth. However, a strain lacking all origins has no apparent defects and grows significantly faster than wild type. Origin-less cells initiate replication at dispersed sites rather than at discrete origins and have an absolute requirement for the recombinase RadA, unlike strains lacking individual origins. Our results demonstrate that homologous recombination alone can efficiently initiate the replication of an entire cellular genome. This raises the question of what purpose replication origins serve and why they have evolved.

Figures

Figure 1
Figure 1. Replication profiles for H. volcanii wild isolate and laboratory strain
(a) Relative copy number plotted against chromosomal co-ordinate for the main chromosome and pHV4 of wild isolate DS2. Circular chromosomes are displayed linearized at position 0, vertical lines mark replication origins. (b) Sequence reads for laboratory strain H26 mapped to the reference genome (DS2). pHV4 shading reflects chromosomal co-ordinate. (c) Sequence reads for H26 mapped to a reconstructed assembly of the main chromosome with pHV4 integrated at ~249 kb. Grey shading from (b) indicates the orientation of pHV4 integration. (d) Integration of pHV4 into the main chromosome of H26.
Figure 2
Figure 2. Characterization of origin deletion strains
(a) Deletion strains were confirmed by hybridization with origin-specific probes (“p” refers to ori-pHV4). (b) Flow cytometry was used to measure DNA content of origin deletion strains, biological replicates are shown; no differences in cell size were observed (data not shown). (c) Pairwise growth competition assays comparing wild-type (H54, bgaHa+) and origin deletion strains. The average and standard error of four independent replicates are plotted.
Figure 3
Figure 3. Origin deletion strain replication profiles
Comparison of replication profiles for (a) laboratory strain (wild type), (b) ΔoriC1, (c) ΔoriC2, (d) ΔoriC3, (e) ΔoriC1,2,3 and (f) ΔoriC1,2,3,pHV4 mutants. Relative copy number for the main chromosome with integrated pHV4 was derived and displayed as in Fig. 1c, dashed lines mark the location of deleted origins.
Figure 4
Figure 4. RadA recombinase is essential in an ΔoriC1,2,3,pHV4 mutant
radA was placed under control of the tryptophan-inducible p.tnaA promoter, in oriC+ and ΔoriC1,2,3,pHV4 strains (H1637 and H1642). The former grows slowly in the absence of tryptophan while the latter is inviable. Absence of tryptophan does not affect the growth of oriC+ and ΔoriC1,2,3,pHV4 control strains (H26 and H1546); the ΔtrpA control strain (H53) is auxotrophic for tryptophan.

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