T
amyE::
PCR-gudB+/ P+-gudBCR
gudBCR
KnS
oriC oriC
5’
amyE::
PCR-gudB+/ P+-gudBCR
gudBCR
oriC
T
KnS 5’ 3’
3’ 5’
3’
5’ 3’
replisome
A
B
Fig. 3.6 Factors involved in TR mutagenesis I
Comparison of mutation frequencies of the natural gudBCR gene encountering a co-directional conflict in GP747 (rocG::Tn10 = rocG-) or of the artificial constructs ∆gudB rocG- PCR-gudB+/∆gudB rocG- P+-gudBCR encountering head-on conflicts (BP404/BP405) harboring secondary deletions of factors putatively involved in TR mutagenesis, determined as described in Ch. 2.2.6.1. The strain was grown to an OD600 of 0.5 to 0.6, washed twice in 1x C-salts, thereby the OD600 was adjusted to 0.4 and 100 µl were used for plating (2.3 ∙ 106 cells). Number of mutants of the respective strains in dependence of time (1, 2, 3, and 4 dpi). A: rocG- (GP747), rocG- ∆rnhB (BP424), rocG- ∆rnhC (BP431), rocG- ∆sbcDC (GP896), rocG- ∆nfo (GP1501). B: rocG- (GP747), rocG- ∆recN (BP629), rocG- ∆recG (BP630), rocG-
Results The mechanism of gudB
CRmutagenesis
Fig. 3.7 Factors involved in TR mutagenesis II
Comparison of mutation frequencies of the artificial constructs ∆gudB rocG- PCR-gudB+/∆gudB rocG- P+-gudBCR encountering head-on conflicts (BP404/BP405) harboring secondary deletions of factors putatively involved in TR mutagenesis, determined as described in Ch. 2.2.6.1. The strain was grown to an OD600 of 0.5 to 0.6, washed twice in 1x C-salts, thereby the OD600 was adjusted to 0.4 and 100 µl were used for plating (2.3 ∙ 106 cells). Number of mutants of the respective strains in dependence of time (1, 2, 3, and 4 dpi). A: PCR -gudB+ (BP404), PCR-gudB+∆rnhB (BP769), PCR-gudB+∆rnhC (BP763), PCR-gudB+∆nfo (BP765), P+-gudBCR (BP405), ∆rnhB P+-gudBCR (BP768),
∆rnhC P+-gudBCR (BP762), ∆nfo P+-gudBCR (BP764). B: PCR-gudB+ (BP404), PCR-gudB+∆recA (BP753), PCR-gudB+∆recJ (BP751), PCR -gudB+∆mfd (BP755), P+-gudBCR (BP405), ∆recA P+-gudBCR (BP754), ∆recJ P+-gudBCR (BP752), ∆mfd P+-gudBCR (BP756). C: PCR-gudB+ (BP404), PCR-gudB+∆recF (BP708), PCR-gudB+∆recR (BP710), PCR-gudB+∆recX (BP712), P+-gudBCR (BP405), ∆recF P+-gudBCR (BP709), ∆recR P+-gudBCR (BP711), ∆recX P+-gudBCR (BP713). D: PCR-gudB+ (BP404), PCR-gudB+∆recN (BP702), PCR-gudB+∆recG (BP704), PCR-gudB+∆recO (BP706), P+-gudBCR (BP405), ∆recN P+-gudBCR (BP703), ∆recG P+-gudBCR (BP705), ∆recO P+-gudBCR (BP707).
gudBCR P+
-10 -35
PCR gudB+
-10 -35
0 500 1000 1500 2000
∆rnhB ∆rnhC ∆nfo ∆rnhB ∆rnhC ∆nfo
Suppressor mutants
0 250 500 750 1000
∆recA ∆recJ ∆mfd ∆recA ∆recJ ∆mfd
Suppressor mutants
0 250 500 750 1000
∆recF ∆recR ∆recX ∆recF ∆recR ∆recX
Suppressor mutants
0 250 500 750 1000
∆recN ∆recG ∆recO ∆recN ∆recG ∆recO
Suppressor mutants
A
B
C
D
1 2 3 4
1 2 2.7 3 4 1 2 2.7 3 4 1 2 2.7 3 4 1 2 2.7 3 4 1 2 2.7 3 4 1 2 2.7 3 4 1 2 2.7 3 4 1 2 2.7 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 dpi
dpi
dpi
dpi
parental strain and the rnhC mutant (Fig. 3.6 A).
However, encountering a head-on conflict, an impact of RNase HII on the mutation frequency of intra- and intergenic TRs is not detectable (Fig.
3.7 A). Interestingly, the RNase HIII seems to be involved in the repair of head-on collisions of intragenic TRs, because a deletion results in a dramatic increase of SMs (Fig. 3.7 A). It makes perfectly sense that neither of the RNase Hs are involved in the repair of intergenic TRs, because RNA-DNA hybrids only occur within genes. It is also likely that there are less conflicts in co-directional orientation compared to head-on orientation and therefore the RNases are less required. As previously stated, RNases H remove RNA-DNA hybrids to ensure a proper replication restart. A deletion of the RNases H would lead to a disruption of this process and the probability of a DNA damage increases. This is reflected by the distinct increase in SMs for the ∆rnhC strain harboring the intragenic TR in head-on direction (Fig. 3.7 A), but not by the decrease in mutation frequency in the ∆rnhB strain harboring the intragenic TR in co-directional orientation.
A detailed description of the repair mechanisms upon collisions of the replication and transcription machinery is given in Ch. 1.4.1.1. To investigate the contribution of the initial recognition of DSB in TR mutagenesis, ∆recN deletion mutants harboring an intragenic TR in co-directional and an inter- and intragenic TR in head-on direction were investigated but no influence was detected (Fig. 3.6 B, Fig. 3.7 D). The main processing of the DSB was investigated using a ∆recJ deletion mutant harboring inter- and intragenic TRs in head-on direction, but did not lead to obviously changed amounts of SMs compared to the parental strains. However, the lack of the RecJ endonuclease might be compensated by the AddAB helicase/nuclease complex. Albeit, the recA gene encodes for the major factor in homologous recombination, its
deletion leads only to a slight decrease in the amount of the SMs in strains harboring an intragenic TR in head-on direction. The deletion of the recO and recR genes had a drastic impact on the amount of SMs in strains harboring an intragenic TR in either direction (Fig. 3.6 B, Fig.
3.7 C, D). If RecO and RecR are required for successful deletion of one TR unit, it is very likely that also RecA is involved in the TR mutagenesis.
In a previous study the impact of different factors building the replication fork in B. subtilis was investigated (Bruand et al., 2001a). There, a kanamycin resistance cassette was artificially inactivated (KnS gene) by a TR and introduced near the native gudBCR gene (Fig. 3.5) in a co-directional manner and no influence of RecA in TR mutagenesis could be detected. However, the deletion of the recA gene in mutants that already have an increased mutation frequency as the dnaD23, the dnaG20, the dnaN5, the dnaX51, or the dnaE1 mutants revealed that these mutants differentially enhance the emergence of SMs in a RecA-dependent or independent manner (Bruand et al., 2001a). Indicated by this study and by the observations made in the previous Ch. 3.1.2, there are several pathways leading to the excision of one TR. Even though the simple deletion of the recA gene has no influence or only a slight influence on TR mutagenesis in this experimental context (Fig. 3.7 B), the general involvement of RecA in TR mutagenesis cannot be ruled out.
To further investigate the importance of RecA in the TR mutagenesis, RecG mediating branch migration, RecF promoting the RecA elongation, and RecX facilitating the disassembly of the RecA filament are tested. However, the three proteins do not have any influence on TR mutagenesis (Fig. 3.6 B, Fig. 3.7 C, D). However, further testing the influence of RecU, which mediates RecA elongation and cleaves Holliday junctions, reveals its involvement in the TR mutagenesis of