The Balance between Recombination Enzymes and Accessory Replicative Helicases in Facilitating Genome Duplication

Abstract

Accessory replicative helicases aid the primary replicative helicase in duplicating protein-bound DNA, especially transcribed DNA. Recombination enzymes also aid genome duplication by facilitating the repair of DNA lesions via strand exchange and also processing of blocked fork DNA to generate structures onto which the replisome can be reloaded. There is significant interplay between accessory helicases and recombination enzymes in both bacteria and lower eukaryotes but how these replication repair systems interact to ensure efficient genome duplication remains unclear. Here, we demonstrate that the DNA content defects of Escherichia coli cells lacking the strand exchange protein RecA are driven primarily by conflicts between replication and transcription, as is the case in cells lacking the accessory helicase Rep. However, in contrast to Rep, neither RecA nor RecBCD, the helicase/exonuclease that loads RecA onto dsDNA ends, is important for maintaining rapid chromosome duplication. Furthermore, RecA and RecBCD together can sustain viability in the absence of accessory replicative helicases but only when transcriptional barriers to replication are suppressed by an RNA polymerase mutation. Our data indicate that the minimisation of replisome pausing by accessory helicases has a more significant impact on successful completion of chromosome duplication than recombination-directed fork repair.

Keywords: RNA polymerase; genome stability; repair; replication.

Figure 1

The chromosome content defects in the absence of Rep and RecA on rich medium are suppressed by an RNA polymerase mutation or by growth on minimal medium. (A) DNA content of the indicated strains grown to mid-logarithmic phase in LB medium as monitored by flow cytometry under run out conditions. The number of chromosome equivalents is indicated below; (B) DNA content of the strains used in (Figure 1A (i–iv)) grown to mid-logarithmic phase in minimal medium as monitored by flow cytometry under run out conditions.

Figure 2

The chromosome content defects of rep and recA mutant cells at mid-logarithmic phase in rich medium are resolved by the time stationary phase is reached. Strains (A–D) are the same as those used in Figure 1A (i–iv).

Figure 3

Chromosome duplication time is extended in rep but not recA or recB cells. (A–D) Flow cytometry profiles of the indicated strains in which initiation of chromosome duplication was synchronised at 42 °C by exploiting the presence of the temperature-sensitive dnaA46 allele. Samples were analysed immediately after shifting the temperature from 42 °C to 30 °C (time 0). Cultures were then returned to 42 °C after 10 min. Samples were removed every 10 min after the temperature downshift. The number of chromosome equivalents is indicated below.

Figure 4

RecA is essential in the absence of Rep and UvrD on rich medium. (A,B) The ability to form colonies in the absence of RecA was monitored in the indicated strains on LB plates containing Xgal and IPTG. The parental strains contain pAM407 (pRC7uvrD) bearing both the uvrD gene and the lac operon and plasmidless cells give rise to white or segregated colonies due to loss of the lac operon. Fractions of white colonies are indicated below each panel and the actual number of white colonies and of total colonies are shown in parentheses.

Figure 5

uvrD recB double mutant cells are viable but have a growth defect. (A–C) The ability of the indicated strains to lose pAM375 (pRC7recB) was monitored on LB Xgal IPTG plates.

Figure 6

RecB is essential in rep uvrD rpoB*35 cells on rich medium. (A–E) Loss of pAM375 (pRC7recB) from the strains indicated was monitored on LB Xgal IPTG.

Figure 7

The requirement for RecBCD and RecA is not alleviated by mutation of recF. (A–D) Loss of pAM406 (pRC7recA, recB) from the strains indicated was monitored on LB Xgal IPTG.

Figure 8

Summary of factors with potential influence on the probability of replisomes completing chromosome duplication and the time needed to do so.