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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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Related in: MedlinePlus

Deficiency of proteins in the RecA-dependent (A) RecBCD and (B) RecFOR pathways for the restart of stalled replication forks results in enhanced instability when GAA is the lagging strand template. Repeat instability was determined for GAA-79 and TTC-79 in recBC, recD, ruvAB, recF, recO and recJ mutants as well as an isogenic wild-type (WT) strain (CL1). GAA-79 is significantly more unstable than TTC-79 in the recBC, ruvAB, recF and recO mutants, but not in the recD and recJ mutants, or in the wild-type strain. In all mutants that showed orientation-dependent instability, instability in the GAA orientation is greater in the mutant versus the isogenic wild-type (P < 0.05 in each case). Error bars depict +/− 2SEM; **P < 0.01; ***P < 0.001; n.s. = not significant.
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Figure 6: Deficiency of proteins in the RecA-dependent (A) RecBCD and (B) RecFOR pathways for the restart of stalled replication forks results in enhanced instability when GAA is the lagging strand template. Repeat instability was determined for GAA-79 and TTC-79 in recBC, recD, ruvAB, recF, recO and recJ mutants as well as an isogenic wild-type (WT) strain (CL1). GAA-79 is significantly more unstable than TTC-79 in the recBC, ruvAB, recF and recO mutants, but not in the recD and recJ mutants, or in the wild-type strain. In all mutants that showed orientation-dependent instability, instability in the GAA orientation is greater in the mutant versus the isogenic wild-type (P < 0.05 in each case). Error bars depict +/− 2SEM; **P < 0.01; ***P < 0.001; n.s. = not significant.

Mentions: Stalling of replication occurs in the (GAA·TTC)n tract specifically when GAA is the lagging strand template (40). Since deficiency of RecA and SSB, proteins that play an important role in the recovery of stalled replication forks, showed increased instability in the GAA orientation, we examined the role of other proteins involved in replication restart. GAA-79 and TTC-79 plasmids were propagated in E. coli strains mutant for recBC (CL3), recD (CL4), ruvAB (CL557), recF (CL579), recO (CL554), and recJ (CL10), and their parental wild-type strain, CL1 (Table 1). The same level of instability was noted in the GAA and TTC orientations in the wild-type CL1 strain (P = 0.39 and P = 0.86 in Figure 6A and B, respectively), which has normal RecA and SSB activities. In the recBC, ruvAB, recF and recO mutants, instability was clearly orientation-dependent, with the (GAA·TTC)79 repeat tract showing significantly more instability when GAA was the lagging strand template (P < 0.01 in each strain; Figure 6A and B). These data indicate that both the RecBCD and RecFOR pathways are required for maintaining stability of the (GAA·TTC)n sequence when GAA is the lagging strand template. Orientation-dependent instability was not seen in the recJ (P = 0.24) and recD (P = 0.42) mutants (Figure 6A and B), indicating that the absence of these proteins is not sufficient to mediate the orientation-dependent instability of the (GAA·TTC)n sequence. In all the mutant strains that showed orientation-dependent instability, invariably the orientation-dependence was due to an increase in the mutation frequency when GAA was the lagging strand template compared with the corresponding wild-type strain (P < 0.05 in each case).Figure 6.


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)

Deficiency of proteins in the RecA-dependent (A) RecBCD and (B) RecFOR pathways for the restart of stalled replication forks results in enhanced instability when GAA is the lagging strand template. Repeat instability was determined for GAA-79 and TTC-79 in recBC, recD, ruvAB, recF, recO and recJ mutants as well as an isogenic wild-type (WT) strain (CL1). GAA-79 is significantly more unstable than TTC-79 in the recBC, ruvAB, recF and recO mutants, but not in the recD and recJ mutants, or in the wild-type strain. In all mutants that showed orientation-dependent instability, instability in the GAA orientation is greater in the mutant versus the isogenic wild-type (P < 0.05 in each case). Error bars depict +/− 2SEM; **P < 0.01; ***P < 0.001; n.s. = not significant.
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Figure 6: Deficiency of proteins in the RecA-dependent (A) RecBCD and (B) RecFOR pathways for the restart of stalled replication forks results in enhanced instability when GAA is the lagging strand template. Repeat instability was determined for GAA-79 and TTC-79 in recBC, recD, ruvAB, recF, recO and recJ mutants as well as an isogenic wild-type (WT) strain (CL1). GAA-79 is significantly more unstable than TTC-79 in the recBC, ruvAB, recF and recO mutants, but not in the recD and recJ mutants, or in the wild-type strain. In all mutants that showed orientation-dependent instability, instability in the GAA orientation is greater in the mutant versus the isogenic wild-type (P < 0.05 in each case). Error bars depict +/− 2SEM; **P < 0.01; ***P < 0.001; n.s. = not significant.
Mentions: Stalling of replication occurs in the (GAA·TTC)n tract specifically when GAA is the lagging strand template (40). Since deficiency of RecA and SSB, proteins that play an important role in the recovery of stalled replication forks, showed increased instability in the GAA orientation, we examined the role of other proteins involved in replication restart. GAA-79 and TTC-79 plasmids were propagated in E. coli strains mutant for recBC (CL3), recD (CL4), ruvAB (CL557), recF (CL579), recO (CL554), and recJ (CL10), and their parental wild-type strain, CL1 (Table 1). The same level of instability was noted in the GAA and TTC orientations in the wild-type CL1 strain (P = 0.39 and P = 0.86 in Figure 6A and B, respectively), which has normal RecA and SSB activities. In the recBC, ruvAB, recF and recO mutants, instability was clearly orientation-dependent, with the (GAA·TTC)79 repeat tract showing significantly more instability when GAA was the lagging strand template (P < 0.01 in each strain; Figure 6A and B). These data indicate that both the RecBCD and RecFOR pathways are required for maintaining stability of the (GAA·TTC)n sequence when GAA is the lagging strand template. Orientation-dependent instability was not seen in the recJ (P = 0.24) and recD (P = 0.42) mutants (Figure 6A and B), indicating that the absence of these proteins is not sufficient to mediate the orientation-dependent instability of the (GAA·TTC)n sequence. In all the mutant strains that showed orientation-dependent instability, invariably the orientation-dependence was due to an increase in the mutation frequency when GAA was the lagging strand template compared with the corresponding wild-type strain (P < 0.05 in each case).Figure 6.

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