## 3 Results  
### Transcription Is a Major Cause of Chromosome Degradation in recA Cells  
One key feature of _E. coli_ cells lacking the strand exchange protein RecA in otherwise unperturbed cells is elevated levels of RecBCD-dependent chromosome degradation [55]. This degradation is manifest as formation of cells with a range of different numbers of chromosome equivalents as detected by flow cytometry [55] (see also Figure 1A (i,iii)). Thus cells require strand exchange for normal chromosomal duplication even in the absence of elevated DNA damage or engineered nucleoprotein barriers. The trigger(s) for this enhanced degradation are unclear and so we tested whether this degradation is attributable to transcription. We employed flow cytometry under run-out conditions to monitor DNA content in cells harbouring either wild type RNA polymerase or a mutant form of the complex resulting from the _rpoB(G1260D)_ allele [6; 89] (Figure 1). _rpoB(G1260D)_ displays the same phenotypes as _rpoB\({}^{*}\)35_ including a stringent phenotype, suppression of _\(\Delta\)rep_\(\Delta\)_uvrD_ double mutant lethality and suppression of chromosome replication defects in \(\Delta\)_rep_ mutant cells [6; 89] Most wild type cells contain 4 chromosome equivalents after run out during logarithmic growth in rich medium in both _rpoB\({}^{+}\)_ and _rpoB(G1260D)_ cells [6; 55] (also compare Figure 1A (i) with Figure 1A (v)). In contrast, \(\Delta\)_rep_rpoB\({}^{+}\) cells lacking the accessory replicative helicase Rep contain 8 chromosomes due to the increased time needed to replicate the chromosome and hence more replication origin firings per cell cycle [6; 29; 90]. _rpoB(G1260D)_ suppresses this \(\Delta\)_rep_ mutant phenotype by reducing replisome pausing, with most \(\Delta\)_rep_rpoB(G1260D)_ cells having 4 rather than 8 chromosomes [6] (see also Figure 1A (v-vi)). We found that _rpoB(G1260D)_ also substantially suppressed the broad spread of chromosome equivalents seen in _recA_ mutant cells (Figure 1A, compare (iii) with (vii)). We also tested cells lacking both Rep and RecA. \(\Delta\)_rep_recA_rpoB\({}^{+}\) cells had a more severe defect in chromosome content as compared with the single mutants (Figure 1A, compare (iv) with (ii) and (iii)). There is therefore significant synergy between Rep and RecA function in maintaining chromosome duplication. However, _rpoB(G1260D)_ still provided partial suppression of this severe defect (Figure 1A, compare (iv) and (viii)). Suppression of chromosome replication defects by _rpoB(G1260D)_ in cells lacking Rep, RecA or both enzymes is consistent with transcription being the primary driver of these defects. Replication-transcription conflicts can also be alleviated by growth of _rpoB\({}^{+}\)_ strains in minimal medium [22; 30]. We tested therefore whether the major differences in DNA content in wild type versus _Aspp_, _recA_ or _Aspp_ _recA_ mutant cells seen in mid-logarithmic cells grown in rich medium were recapitulated in minimal medium. We found that the majority of _rpoB\({}^{+}\)_ cells either with or without _Aspp_ and/or _recA_ mutant alleles contained 2 chromosome equivalents when grown to mid-logarithmic phase in minimal medium (Figure 1B (i-iv)). Restricting growth rate reduces therefore the chromosomal defects caused by the absence of Rep and/or RecA (compare Figure 1A (i-iv) with Figure 1B (i-iv)) supporting our conclusion that transcription is a major cause of the perturbed chromosome content observed in the absence of Rep and/or RecA. 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.