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Deficiency of RecA-dependent RecFOR and RecBCD pathways causes increased instability of the (GAA*TTC)n sequence when GAA is the lagging strand template.

Pollard LM, Chutake YK, Rindler PM, Bidichandani SI - Nucleic Acids Res. (2007)

Bottom Line: We also found the same orientation-dependent increase in instability in a RecA+ temperature-sensitive E. coli SSB mutant strain (ssb-1).Consistent with this hypothesis, we noted significantly increased instability when GAA was the lagging strand template in strains that were deficient in components of the RecFOR and RecBCD pathways.Our data implicate defective processing of stalled replication forks as a mechanism for genetic instability of the (GAA*TTC)n sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.

ABSTRACT
The most common mutation in Friedreich ataxia is an expanded (GAA*TTC)n sequence, which is highly unstable in human somatic cells and in the germline. The mechanisms responsible for this genetic instability are poorly understood. We previously showed that cloned (GAA*TTC)n sequences replicated in Escherichia coli are more unstable when GAA is the lagging strand template, suggesting erroneous lagging strand synthesis as the likely mechanism for the genetic instability. Here we show that the increase in genetic instability when GAA serves as the lagging strand template is seen in RecA-deficient but not RecA-proficient strains. We also found the same orientation-dependent increase in instability in a RecA+ temperature-sensitive E. coli SSB mutant strain (ssb-1). Since stalling of replication is known to occur within the (GAA*TTC)n sequence when GAA is the lagging strand template, we hypothesized that genetic stability of the (GAA*TTC)n sequence may require efficient RecA-dependent recombinational restart of stalled replication forks. Consistent with this hypothesis, we noted significantly increased instability when GAA was the lagging strand template in strains that were deficient in components of the RecFOR and RecBCD pathways. Our data implicate defective processing of stalled replication forks as a mechanism for genetic instability of the (GAA*TTC)n sequence.

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The inability to induce the SOS response is not associated with enhanced instability in the GAA orientation. Repeat instability was determined for GAA-79 and TTC-79 in the lexA1(Ind−) mutant (CL103) as well as an isogenic wild-type (WT) strain (CL43). GAA-79 is slightly more unstable than TTC-79 in the lexA1(Ind−) mutant, however, there was no difference in instability in the GAA or TTC orientations when compared with the wild-type strain (P = 0.14 and P = 0.98, respectively). Error bars depict +/− 2SEM; *P < 0.05; n.s. = not significant.
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Figure 7: The inability to induce the SOS response is not associated with enhanced instability in the GAA orientation. Repeat instability was determined for GAA-79 and TTC-79 in the lexA1(Ind−) mutant (CL103) as well as an isogenic wild-type (WT) strain (CL43). GAA-79 is slightly more unstable than TTC-79 in the lexA1(Ind−) mutant, however, there was no difference in instability in the GAA or TTC orientations when compared with the wild-type strain (P = 0.14 and P = 0.98, respectively). Error bars depict +/− 2SEM; *P < 0.05; n.s. = not significant.

Mentions: The observation of increased instability when GAA serves as the lagging strand template in the absence of RecA, SSB and RecFOR, suggested that the absence of one or more proteins induced in the SOS response may be involved in mediating the orientation-dependent instability. We therefore propagated GAA-79 and TTC-79 in the lexA1(Ind−) mutant (CL103), which is unable to turn on the SOS response because it has a non-cleavable LexA, and CL43, its isogenic normal counterpart (Figure 7) (Table 1). There was a slightly higher instability with GAA-79 versus TTC-79 in the lexA1(Ind−) mutant (P < 0.05). However, comparison of the instability in the mutant versus wild-type strains showed that this observation is unlikely to be biologically significant, since there was no difference in instability between the two strains in either the GAA or TTC orientations (P = 0.14 and P = 0.98, respectively) (Figure 7). These data therefore indicate that the effect of increased instability in the GAA orientation seen in the RecA, SSB and RecFOR mutants is unlikely to be due to deficient induction of the SOS response.Figure 7.


Deficiency of RecA-dependent RecFOR and RecBCD pathways causes increased instability of the (GAA*TTC)n sequence when GAA is the lagging strand template.

Pollard LM, Chutake YK, Rindler PM, Bidichandani SI - Nucleic Acids Res. (2007)

The inability to induce the SOS response is not associated with enhanced instability in the GAA orientation. Repeat instability was determined for GAA-79 and TTC-79 in the lexA1(Ind−) mutant (CL103) as well as an isogenic wild-type (WT) strain (CL43). GAA-79 is slightly more unstable than TTC-79 in the lexA1(Ind−) mutant, however, there was no difference in instability in the GAA or TTC orientations when compared with the wild-type strain (P = 0.14 and P = 0.98, respectively). Error bars depict +/− 2SEM; *P < 0.05; n.s. = not significant.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2175318&req=5

Figure 7: The inability to induce the SOS response is not associated with enhanced instability in the GAA orientation. Repeat instability was determined for GAA-79 and TTC-79 in the lexA1(Ind−) mutant (CL103) as well as an isogenic wild-type (WT) strain (CL43). GAA-79 is slightly more unstable than TTC-79 in the lexA1(Ind−) mutant, however, there was no difference in instability in the GAA or TTC orientations when compared with the wild-type strain (P = 0.14 and P = 0.98, respectively). Error bars depict +/− 2SEM; *P < 0.05; n.s. = not significant.
Mentions: The observation of increased instability when GAA serves as the lagging strand template in the absence of RecA, SSB and RecFOR, suggested that the absence of one or more proteins induced in the SOS response may be involved in mediating the orientation-dependent instability. We therefore propagated GAA-79 and TTC-79 in the lexA1(Ind−) mutant (CL103), which is unable to turn on the SOS response because it has a non-cleavable LexA, and CL43, its isogenic normal counterpart (Figure 7) (Table 1). There was a slightly higher instability with GAA-79 versus TTC-79 in the lexA1(Ind−) mutant (P < 0.05). However, comparison of the instability in the mutant versus wild-type strains showed that this observation is unlikely to be biologically significant, since there was no difference in instability between the two strains in either the GAA or TTC orientations (P = 0.14 and P = 0.98, respectively) (Figure 7). These data therefore indicate that the effect of increased instability in the GAA orientation seen in the RecA, SSB and RecFOR mutants is unlikely to be due to deficient induction of the SOS response.Figure 7.

Bottom Line: We also found the same orientation-dependent increase in instability in a RecA+ temperature-sensitive E. coli SSB mutant strain (ssb-1).Consistent with this hypothesis, we noted significantly increased instability when GAA was the lagging strand template in strains that were deficient in components of the RecFOR and RecBCD pathways.Our data implicate defective processing of stalled replication forks as a mechanism for genetic instability of the (GAA*TTC)n sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.

ABSTRACT
The most common mutation in Friedreich ataxia is an expanded (GAA*TTC)n sequence, which is highly unstable in human somatic cells and in the germline. The mechanisms responsible for this genetic instability are poorly understood. We previously showed that cloned (GAA*TTC)n sequences replicated in Escherichia coli are more unstable when GAA is the lagging strand template, suggesting erroneous lagging strand synthesis as the likely mechanism for the genetic instability. Here we show that the increase in genetic instability when GAA serves as the lagging strand template is seen in RecA-deficient but not RecA-proficient strains. We also found the same orientation-dependent increase in instability in a RecA+ temperature-sensitive E. coli SSB mutant strain (ssb-1). Since stalling of replication is known to occur within the (GAA*TTC)n sequence when GAA is the lagging strand template, we hypothesized that genetic stability of the (GAA*TTC)n sequence may require efficient RecA-dependent recombinational restart of stalled replication forks. Consistent with this hypothesis, we noted significantly increased instability when GAA was the lagging strand template in strains that were deficient in components of the RecFOR and RecBCD pathways. Our data implicate defective processing of stalled replication forks as a mechanism for genetic instability of the (GAA*TTC)n sequence.

Show MeSH
Related in: MedlinePlus