Localization of an accessory helicase at the replisome is critical in sustaining efficient genome duplication

Abstract

Genome duplication requires accessory helicases to displace proteins ahead of advancing replication forks. Escherichia coli contains three helicases, Rep, UvrD and DinG, that might promote replication of protein-bound DNA. One of these helicases, Rep, also interacts with the replicative helicase DnaB. We demonstrate that Rep is the only putative accessory helicase whose absence results in an increased chromosome duplication time. We show also that the interaction between Rep and DnaB is required for Rep to maintain rapid genome duplication. Furthermore, this Rep-DnaB interaction is critical in minimizing the need for both recombinational processing of blocked replication forks and replisome reassembly, indicating that colocalization of Rep and DnaB minimizes stalling and subsequent inactivation of replication forks. These data indicate that E. coli contains only one helicase that acts as an accessory motor at the fork in wild-type cells, that such an activity is critical for the maintenance of rapid genome duplication and that colocalization with the replisome is crucial for this function. Given that the only other characterized accessory motor, Saccharomyces cerevisiae Rrm3p, associates physically with the replisome, our demonstration of the functional importance of such an association indicates that colocalization may be a conserved feature of accessory replicative motors.

Figure 1.

The impact of Rep, UvrD and DinG on chromosome duplication time. (A–F) Flow cytometry profiles of the indicated strains, all bearing the temperature sensitive dnaA46 allele. Cell samples were removed for analysis immediately prior to shifting the cultures from 42°C to 30°C (designated 0 min). After 10 min at 30°C the cultures were returned to 42°C. Samples were then removed at 10-min intervals after the temperature downshift. The positions of 1 and 2 chromosome equivalents are indicated.

Figure 2.

Increased chromosome duplication time in the absence of Rep is not due to the absence of PriC-directed replisome reassembly. (A–C) Flow cytometry profiles of the indicated dnaA5-containing strains. Samples were removed just before shifting the cells to 30°C (time zero) and every 10 min thereafter.

Figure 3.

The Rep–DnaB interaction promotes viability of ΔuvrD cells in a transcription-dependent manner. (A) The ability of the indicated strains to form colonies in the absence of plasmid-encoded UvrD, as indicated by the formation of white colonies in the presence of Xgal and IPTG, was monitored under rapid growth conditions (LB) and under restricted growth conditions (MA, minimal agar). Numbers under each panel refer to the fraction of white colonies obtained, whilst the numbers in parentheses indicate the actual number of white colonies and total number of colonies. (B) (i) Growth rates of ΔuvrD rep⁺ (N7912), ΔuvrD repΔC33 (N7913) and ΔuvrD Δrep (N6644), all lacking pRC7 encoding UvrD, upon transfer of cells from minimal medium into LB. (ii) Median lengths of cells grown in minimal medium immediately prior to transfer into LB, and after 3 h in LB. (C) Retention or loss of pRC7-encoded UvrD in (i) ΔuvrD rep⁺ and (ii) ΔuvrD repΔC33 strains bearing rpoB*35 was monitored on LB plates containing Xgal and IPTG.

Figure 4.

The Rep–DnaB interaction minimizes the need for blocked fork processing and replication restart. (A) Retention or loss of pRC7-encoded Rep in rec⁺ and recB strains bearing chromosomal rep⁺ or repΔC33 alleles monitored on LB plates containing Xgal and IPTG. The inset panel in (iv) is an expanded view of blue and white colonies, illustrating the formation of micro-colonies alongside the large plasmid-containing blue colonies. (B) Retention or loss of pRC7-encoded PriA in priA⁺ and ΔpriA strains bearing chromosomal rep⁺ or repΔC33 alleles monitored on LB containing Xgal and IPTG.

Figure 5.

The Rep–DnaB interaction does not alter the mean integral speed of the replisome. Chromosome duplication was monitored by flow cytometry in dnaA46 strains bearing rep alleles encoding (A) wild-type Rep and (B) RepK28R. Time zero indicates samples removed after 2 h at 42°C immediately prior to shifting the cultures to 30°C. Positions of 1 and 2 chromosome equivalents are indicated.