## 3 Results
### Both RecA and RecBCD Are Needed in the Absence of Accessory Helicase Activity  
The above data suggest Rep rather than RecA plays the dominant role in ensuring rapid genome duplication. It is clear, though, that transcription is a shared source of replicative defects in cells deficient in either Rep or RecA (Figure 1). However, the viability of _rep recA_ double mutant cells [71] argues against a requirement for either Rep or RecA to overcome transcriptional barriers to replication. Interpretation of _rep recA_ double mutant viability is complicated, though, since UvrD compensates partially for the absence of Rep accessory helicase function [22; 30]. The requirement for RecA was tested therefore in the absence of both Rep and UvrD by using a _\(\Delta\)rep_\(\Delta\)_uvrD_ double mutant strain rendered viable by the _rpoB*35_ allele via a reduction in replication-transcription conflicts [22; 79; 42]. A plasmid loss assay was employed in which retention of a highly unstable complementing plasmid can be monitored by blue/white screening [87]. _\(\Delta\)rep_\(\Delta\)_uvrD_ _rpoB*35_ cells can lose pRC7_uvrD_ on LB as indicated by the formation of white plasmidless colonies [22] (see also Figure 4A). In contrast, _\(\Delta\)rep_\(\Delta\)_uvrD_ _rpoB*35 recA_ cells could not lose pRC7_uvrD_ on LB indicating that RecA is essential for viability in a _\(\Delta\)_rep_\(\Delta\)_uvrD_ _rpoB*35_ strain under rapid growth conditions (Figure 4, compare B with A). Thus even when transcriptional barriers to replication are reduced by the _rpoB*35_ allele there remains a requirement for either accessory helicase function or RecA. The corollary of _\(\Delta\)_uvrD_ _rpoB*35 recA_ in viability is that RecBCD is unable to maintain viability without RecA in this context. This requirement for RecA is in contrast to the viability of _rep recA_ double mutant cells versus the inviability of _rep recB_ double mutants in a _rpoB+_ background [71]. This differential requirement in _uvrD+ rep-_ cells reflects the generation of double-stranded DNA ends by regression of blocked replication forks and the need for RecBCD to process these ends [58]. Processing can occur either by loading of RecA followed by strand exchange or, in the absence of RecA, RecBCD-catalysed degradation of the dsDNA end to regenerate a fork structure [58]. The viability of _uvrD recB_ double mutant strains is less certain. Absence of UvrD-catalysed removal of RecFOR-loaded RecA from blocked forks may lead to an increased need for RecBCD-dependent repair of dsDNA ends [84]. Some reports indicate reduced viability of _uvrD recB_ mutant strains [91] whereas others report inviability [92; 93]. We assayed the viability of _\(\Delta\)_uvrD recB-_rpoB+_ cells by analysing their ability to lose pRC7_recB_. _\(\Delta\)_uvrD_ _recB-_rpoB+_ cells could generate white colonies on rich medium in contrast to _\(\Delta\)_rep_\(\Delta\  
white colony formation was lower and white colony sizes much smaller than with \(uvrD^{+}\)_recB\({}^{-}\)_rpoB\({}^{+}\)_cells (Figure 5, compare B with A). Thus, in this strain, background cells can survive without both UvrD and RecBCD but growth is impaired. _rpo_ mutations that suppress \(\Delta\)_rep_\(\Delta\)_uvrD_ double mutant lethality are unable to suppress \(\Delta\)_rep_\(recB\) double mutant lethality [78]. Similarly, reduction of transcription-driven replicative barriers using _rpoB*35_ did not improve the viability of either \(\Delta\)_rep_\(recB\) or \(\Delta\)_uvrD_\(recB\) double mutant strains (compare Figure 6C with Figure 5B; Figure 6D with Figure 5C). _Arp_\(\Delta\)_uvrD_rpoB*35_\(recB^{-}\) was also inviable (Figure 6E), as expected given the growth defects of single _rep_ and _uvrD_ mutants [71] (Figure 6C,D). It was possible that UvrD not being available to abort RecFOR-directed loading of RecA onto blocked replication forks [81; 84] contributed to \(\Delta\)_rep_\(\Delta\)_uvrD_\(rpoB*35\)_recB_ inviability.