Spontaneous DNA breakage in single living Escherichia coli cells

Abstract

Spontaneous DNA breakage is predicted to be a frequent, inevitable consequence of DNA replication and is thought to underlie much of the genomic change that fuels cancer and evolution [1][2][3] . Despite its importance, there has been little direct measurement of the amounts, types, sources and fates of spontaneous DNA lesions in living cells. We present a direct, sensitive flow cytometric assay in single living Escherichia coli cells for DNA lesions capable of inducing the SOS DNA damage response, and we report its use in quantification of spontaneous DNA double-strand breaks (DSBs). We report efficient detection of single chromosomal DSBs and rates of spontaneous breakage ~ 20-to 100-fold lower than predicted. In addition, we implicate DNA replication in the origin of spontaneous DSBs with the finding of fewer spontaneous DSBs in a mutant with altered DNA polymerase III. The data imply that spontaneous DSBs induce genomic changes and instability 20-100 times more potently than previously appreciated. Finally, FACS demonstrated two main cell fates after spontaneous DNA damage: viability with or without resumption of proliferation.DNA DSBs are important instigators of genomic instability that provoke mutation, genome rearrangement and chromosome aberrations important in evolution, oncogenesis and genetic disease [3][4][5] . Frequent DSBs are thought to arise spontaneously and to be repaired accurately when normal DNA replication encounters damage from endogenous causes [1][2][3] . Indirect estimates of rates of spontaneous DSB formation have been derived from inviability, chromosome loss or cytogenetic phenotypes of cells lacking DNA repair proteins, some of which affect processes other than repair 1-3 . These estimates are ambiguous, ranging from 0.2-1 per genome replication in Escherichia coli 1,2 and 50 per human genome replication 3 , and the extent to which the phenotypes underlying these estimates are attributed to DSBs

Roles of Nucleoid-Associated Proteins in Stress-Induced Mutagenic Break Repair in StarvingEscherichia coli

“…Loss of CbpA did not affect the frequencies of spontaneous DNA damage or SOS induction, as shown with the flow cytometric assay of Pennington and Rosenberg (2007) for fluorescent SOS-induced cells ( Figure 3D) carrying a chromosomal SOS-regulated PsulA-gfp transgene. Isogenic PsulA-gfp cells with the SOS-blocking lexA3(Ind 2 ) mutation demonstrate the SOS dependence of fluorescence in this assay ( Figure 3D) (Pennington and Rosenberg 2007).…”

“…All strains used for SOS induction determination carried the SOS-regulated PsulA-gfp allele (Pennington and Rosenberg 2007). PsulA-gfp is inserted in a nongenic chromosomal site in sulA + cells (Pennington and Rosenberg 2007).…”

“…PsulA-gfp is inserted in a nongenic chromosomal site in sulA + cells (Pennington and Rosenberg 2007). Five-milliliter cultures of isogenic SMR6039 (SOS-proficient control), SMR6178 [lexA3(Ind) SOS-defective], and an NAP deletion mutant were grown for 3 days at 32°in M9 minimal glycerol medium as in MBR starvation assay conditions.…”

“…(1) a quantitative P1 phage-mediated transduction test of HR proficiency at DSB ends, a proxy for DSB repair by HR, (2) sensitivity to UV light, which requires functional DSB repair (Khan and Kuzminov 2012), (3) a flow-cytometric assay for the ability to induce the SOS response, which uses an SOSregulated promoter fused to gfp (Pennington and Rosenberg 2007), and (4) an assay for genes that promote MBR by contributing to induction of the RpoS general stress response showing requirements for NAPs for MBR and summary of MBR rates from three experiments (right) (mean rates 6 SEM) compared with the wild-type protein. Deletion of hns, fis, cbpA, and cspE reduced MBR rates (P = 0.005, 0.03, 0.03, and 0.049, respectively, Student's two-tailed t-test), whereas Ddps increased the mutation rate.…”

Completion of DNA replication in Escherichia coli

“…1 C and F demonstrate, the proportion of broken, linear plasmids is actually lower in recBC or recD cultures relative to wild-type or other recombination mutants. Additionally, double-strand breaks are estimated to arise in vivo at frequencies ranging from 0.01 to 1 break per 4.5 Mb of replicated genome (30), making it unlikely that these account for the instability of a 4.5-kb plasmid. Finally, plasmids remain stable and replicate normally in recA mutants, which are defective in all homologous recombination and RecBCDmediated double-strand break repair ( Fig.…”