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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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Reduction in SSB activity causes a length-dependent increase in (GAA·TTC)n instability. (A) Representative agarose gels showing PCR products generated from colonies containing plasmids with the indicated lengths of (GAA·TTC)n repeat tracts in the GAA orientation in C600 (wild-type (WT) for SSB), and in the isogenic temperature sensitive ssb-1 mutant, RM121. Arrowheads indicate the position of the respective full-length repeats. (B) (GAA·TTC)n instability is length-dependent in both strains, however, the GAA-41 and GAA-79 repeat tracts are significantly more unstable in RM121 (P<0.001 and P<0.01 for GAA-41 and GAA-79, respectively). Error bars depict +/− 2SEM. (C) Repeat instability was determined for GAA-41 propagated in the ssb-1 mutant and C600 strains at the permissive versus non-permissive temperatures (25°C (white bars) versus 37°C (black bars)). Instability of GAA-41 was significantly higher at 37°C in ssb-1 compared with 25°C. Increase in the temperature per se did not affect instability since C600 showed the same level of instability at both temperatures. The increased instability in the ssb-1 mutant versus wild-type even at the permissive temperature is likely due to leaky expression of the mutant phenotype caused by a partial deficiency of SSB. Error bars depict +/− 2SEM; **P < 0.01; n.s. = not significant.
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Figure 4: Reduction in SSB activity causes a length-dependent increase in (GAA·TTC)n instability. (A) Representative agarose gels showing PCR products generated from colonies containing plasmids with the indicated lengths of (GAA·TTC)n repeat tracts in the GAA orientation in C600 (wild-type (WT) for SSB), and in the isogenic temperature sensitive ssb-1 mutant, RM121. Arrowheads indicate the position of the respective full-length repeats. (B) (GAA·TTC)n instability is length-dependent in both strains, however, the GAA-41 and GAA-79 repeat tracts are significantly more unstable in RM121 (P<0.001 and P<0.01 for GAA-41 and GAA-79, respectively). Error bars depict +/− 2SEM. (C) Repeat instability was determined for GAA-41 propagated in the ssb-1 mutant and C600 strains at the permissive versus non-permissive temperatures (25°C (white bars) versus 37°C (black bars)). Instability of GAA-41 was significantly higher at 37°C in ssb-1 compared with 25°C. Increase in the temperature per se did not affect instability since C600 showed the same level of instability at both temperatures. The increased instability in the ssb-1 mutant versus wild-type even at the permissive temperature is likely due to leaky expression of the mutant phenotype caused by a partial deficiency of SSB. Error bars depict +/− 2SEM; **P < 0.01; n.s. = not significant.

Mentions: Since SSB is known to play an important role in RecA activity, we also examined the effect of reduced SSB activity on (GAA·TTC)n instability. Constructs containing various lengths of the (GAA·TTC)n sequence, cloned in the GAA orientation relative to the origin of replication (GAA-21, GAA-41 and GAA-79) (Figure 1), were propagated in the wild-type C600 strain and its derivative, RM121, which contains a temperature-sensitive SSB mutation (ssb-1) (47) (Table 1). Instability was measured by culturing both strains at the non-permissive temperature (37°C), at which there is greatly reduced SSB activity in the RM121 strain (53). Instability of the (GAA·TTC)n sequence was found to be length-dependent in the ssb-1 strain at the non-permissive temperature, and the repeat tracts were significantly more unstable in the ssb-1 strain than in the parental wild-type strain (P < 0.001 for GAA-41 and P = 0.02 for GAA-79) (Figure 4A and B), indicating that reduction of SSB activity increases (GAA·TTC)n instability. The much higher level of instability of the GAA-79 construct propagated in RM121 at the non-permissive temperature (which made it very difficult to quantify accurately; compare Figures 2B and 4A), indicates that reduction in SSB activity produces a more severe destabilization of the (GAA·TTC)n repeat sequence compared with the deficiency of RecA. Therefore, further detailed characterization of the effect of reduced SSB activity was performed using the GAA-41 construct. Propagation of the GAA-41 construct in ssb-1 and the wild-type control at 25 and 37°C, showed a significantly higher level of instability in the ssb-1 strain at the non-permissive temperature (Figure 4C). Although GAA-41 was significantly more unstable in ssb-1 than in the wild-type at both 25°C and 37°C (P = 0.007 and P < 0.001, respectively), no difference in instability was observed in the wild-type strain grown at 25°C and 37°C (P = 0.95). This demonstrates that the specific reduction in SSB activity at the non-permissive temperature is responsible for the increased instability of GAA-41 in ssb-1, and the latter is not simply due to increase in temperature.Figure 4.


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)

Reduction in SSB activity causes a length-dependent increase in (GAA·TTC)n instability. (A) Representative agarose gels showing PCR products generated from colonies containing plasmids with the indicated lengths of (GAA·TTC)n repeat tracts in the GAA orientation in C600 (wild-type (WT) for SSB), and in the isogenic temperature sensitive ssb-1 mutant, RM121. Arrowheads indicate the position of the respective full-length repeats. (B) (GAA·TTC)n instability is length-dependent in both strains, however, the GAA-41 and GAA-79 repeat tracts are significantly more unstable in RM121 (P<0.001 and P<0.01 for GAA-41 and GAA-79, respectively). Error bars depict +/− 2SEM. (C) Repeat instability was determined for GAA-41 propagated in the ssb-1 mutant and C600 strains at the permissive versus non-permissive temperatures (25°C (white bars) versus 37°C (black bars)). Instability of GAA-41 was significantly higher at 37°C in ssb-1 compared with 25°C. Increase in the temperature per se did not affect instability since C600 showed the same level of instability at both temperatures. The increased instability in the ssb-1 mutant versus wild-type even at the permissive temperature is likely due to leaky expression of the mutant phenotype caused by a partial deficiency of SSB. Error bars depict +/− 2SEM; **P < 0.01; n.s. = not significant.
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Figure 4: Reduction in SSB activity causes a length-dependent increase in (GAA·TTC)n instability. (A) Representative agarose gels showing PCR products generated from colonies containing plasmids with the indicated lengths of (GAA·TTC)n repeat tracts in the GAA orientation in C600 (wild-type (WT) for SSB), and in the isogenic temperature sensitive ssb-1 mutant, RM121. Arrowheads indicate the position of the respective full-length repeats. (B) (GAA·TTC)n instability is length-dependent in both strains, however, the GAA-41 and GAA-79 repeat tracts are significantly more unstable in RM121 (P<0.001 and P<0.01 for GAA-41 and GAA-79, respectively). Error bars depict +/− 2SEM. (C) Repeat instability was determined for GAA-41 propagated in the ssb-1 mutant and C600 strains at the permissive versus non-permissive temperatures (25°C (white bars) versus 37°C (black bars)). Instability of GAA-41 was significantly higher at 37°C in ssb-1 compared with 25°C. Increase in the temperature per se did not affect instability since C600 showed the same level of instability at both temperatures. The increased instability in the ssb-1 mutant versus wild-type even at the permissive temperature is likely due to leaky expression of the mutant phenotype caused by a partial deficiency of SSB. Error bars depict +/− 2SEM; **P < 0.01; n.s. = not significant.
Mentions: Since SSB is known to play an important role in RecA activity, we also examined the effect of reduced SSB activity on (GAA·TTC)n instability. Constructs containing various lengths of the (GAA·TTC)n sequence, cloned in the GAA orientation relative to the origin of replication (GAA-21, GAA-41 and GAA-79) (Figure 1), were propagated in the wild-type C600 strain and its derivative, RM121, which contains a temperature-sensitive SSB mutation (ssb-1) (47) (Table 1). Instability was measured by culturing both strains at the non-permissive temperature (37°C), at which there is greatly reduced SSB activity in the RM121 strain (53). Instability of the (GAA·TTC)n sequence was found to be length-dependent in the ssb-1 strain at the non-permissive temperature, and the repeat tracts were significantly more unstable in the ssb-1 strain than in the parental wild-type strain (P < 0.001 for GAA-41 and P = 0.02 for GAA-79) (Figure 4A and B), indicating that reduction of SSB activity increases (GAA·TTC)n instability. The much higher level of instability of the GAA-79 construct propagated in RM121 at the non-permissive temperature (which made it very difficult to quantify accurately; compare Figures 2B and 4A), indicates that reduction in SSB activity produces a more severe destabilization of the (GAA·TTC)n repeat sequence compared with the deficiency of RecA. Therefore, further detailed characterization of the effect of reduced SSB activity was performed using the GAA-41 construct. Propagation of the GAA-41 construct in ssb-1 and the wild-type control at 25 and 37°C, showed a significantly higher level of instability in the ssb-1 strain at the non-permissive temperature (Figure 4C). Although GAA-41 was significantly more unstable in ssb-1 than in the wild-type at both 25°C and 37°C (P = 0.007 and P < 0.001, respectively), no difference in instability was observed in the wild-type strain grown at 25°C and 37°C (P = 0.95). This demonstrates that the specific reduction in SSB activity at the non-permissive temperature is responsible for the increased instability of GAA-41 in ssb-1, and the latter is not simply due to increase in temperature.Figure 4.

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