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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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RecA deficiency causes orientation-dependent (GAA·TTC)n instability due to increased instability when GAA is the lagging strand template. (A) Repeat instability was significantly enhanced for GAA-79 (white bars) versus TTC-79 (black bars) propagated in three RecA-deficient strains (DH5α, HB101, Top10), but no such orientation-dependence was seen in the three RecA-proficient strains (C600, KA796, KH1370). (B) Representative gels showing enhanced repeat instability for GAA-79 versus TTC-79 propagated in M152 (RecA-deficient) but not in the isogenic strain MM28 (RecA-proficient). Arrowheads indicate the position of the full-length (GAA·TTC)79 repeat. (C) Instability was significantly enhanced for GAA-79 versus TTC-79 in M152 (RecA-deficient) but not in MM28 (RecA-proficient). Error bars depict +/− 2SEM; **P < 0.01; ***P < 0.001; n.s. = not significant.
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Figure 2: RecA deficiency causes orientation-dependent (GAA·TTC)n instability due to increased instability when GAA is the lagging strand template. (A) Repeat instability was significantly enhanced for GAA-79 (white bars) versus TTC-79 (black bars) propagated in three RecA-deficient strains (DH5α, HB101, Top10), but no such orientation-dependence was seen in the three RecA-proficient strains (C600, KA796, KH1370). (B) Representative gels showing enhanced repeat instability for GAA-79 versus TTC-79 propagated in M152 (RecA-deficient) but not in the isogenic strain MM28 (RecA-proficient). Arrowheads indicate the position of the full-length (GAA·TTC)79 repeat. (C) Instability was significantly enhanced for GAA-79 versus TTC-79 in M152 (RecA-deficient) but not in MM28 (RecA-proficient). Error bars depict +/− 2SEM; **P < 0.01; ***P < 0.001; n.s. = not significant.

Mentions: To investigate the effect of RecA status on (GAA·TTC)n instability, GAA-79 and TTC-79, constructs containing a (GAA·TTC)79 repeat sequence in pUC19 in both orientations relative to the pMB1 origin of replication (Figure 1), were propagated in three RecA-deficient (DH5α, Top10, HB101) and three RecA-proficient strains (C600, KA796, KH1370) (Table 1). Colony PCR was used to visualize products of individual replication events in order to detect repeat instability. GAA-79 was significantly more unstable than TTC-79 in all three recA mutant strains (Figure 2A). In contrast, all three RecA+ strains did not show a difference in the level of instability between GAA-79 and TTC-79 (Figure 2A). These data suggest that deficiency of RecA causes an orientation-dependent instability of the (GAA·TTC)79 sequence. However, the differing genetic backgrounds of the various strains (Table 1) resulted in widely varying absolute levels of instability. Also, it was not possible to determine if the orientation-dependence stemmed from increased instability in the GAA orientation, decreased instability in the TTC orientation, or both. Therefore, to further investigate the role of RecA status and to control for the confounding effects of varying genetic backgrounds, GAA-79 and TTC-79 plasmids were propagated in a set of isogenic strains, M152 (recA) and MM28 (wild-type) (Table 1). Again, GAA-79 was significantly more unstable than TTC-79 in the recA strain (Figure 2B and C), indicating that the orientation-dependent instability is specifically due to the deficiency of RecA. Furthermore, since GAA-79 was significantly more unstable than TTC-79 in the recA strain, and there was no difference in the instability of TTC-79 between the two strains (P = 0.163; Figure 2C), it indicates that the orientation-dependent instability in the absence of RecA is due to an increase in instability when GAA is the lagging strand template.Figure 2.


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)

RecA deficiency causes orientation-dependent (GAA·TTC)n instability due to increased instability when GAA is the lagging strand template. (A) Repeat instability was significantly enhanced for GAA-79 (white bars) versus TTC-79 (black bars) propagated in three RecA-deficient strains (DH5α, HB101, Top10), but no such orientation-dependence was seen in the three RecA-proficient strains (C600, KA796, KH1370). (B) Representative gels showing enhanced repeat instability for GAA-79 versus TTC-79 propagated in M152 (RecA-deficient) but not in the isogenic strain MM28 (RecA-proficient). Arrowheads indicate the position of the full-length (GAA·TTC)79 repeat. (C) Instability was significantly enhanced for GAA-79 versus TTC-79 in M152 (RecA-deficient) but not in MM28 (RecA-proficient). Error bars depict +/− 2SEM; **P < 0.01; ***P < 0.001; n.s. = not significant.
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Figure 2: RecA deficiency causes orientation-dependent (GAA·TTC)n instability due to increased instability when GAA is the lagging strand template. (A) Repeat instability was significantly enhanced for GAA-79 (white bars) versus TTC-79 (black bars) propagated in three RecA-deficient strains (DH5α, HB101, Top10), but no such orientation-dependence was seen in the three RecA-proficient strains (C600, KA796, KH1370). (B) Representative gels showing enhanced repeat instability for GAA-79 versus TTC-79 propagated in M152 (RecA-deficient) but not in the isogenic strain MM28 (RecA-proficient). Arrowheads indicate the position of the full-length (GAA·TTC)79 repeat. (C) Instability was significantly enhanced for GAA-79 versus TTC-79 in M152 (RecA-deficient) but not in MM28 (RecA-proficient). Error bars depict +/− 2SEM; **P < 0.01; ***P < 0.001; n.s. = not significant.
Mentions: To investigate the effect of RecA status on (GAA·TTC)n instability, GAA-79 and TTC-79, constructs containing a (GAA·TTC)79 repeat sequence in pUC19 in both orientations relative to the pMB1 origin of replication (Figure 1), were propagated in three RecA-deficient (DH5α, Top10, HB101) and three RecA-proficient strains (C600, KA796, KH1370) (Table 1). Colony PCR was used to visualize products of individual replication events in order to detect repeat instability. GAA-79 was significantly more unstable than TTC-79 in all three recA mutant strains (Figure 2A). In contrast, all three RecA+ strains did not show a difference in the level of instability between GAA-79 and TTC-79 (Figure 2A). These data suggest that deficiency of RecA causes an orientation-dependent instability of the (GAA·TTC)79 sequence. However, the differing genetic backgrounds of the various strains (Table 1) resulted in widely varying absolute levels of instability. Also, it was not possible to determine if the orientation-dependence stemmed from increased instability in the GAA orientation, decreased instability in the TTC orientation, or both. Therefore, to further investigate the role of RecA status and to control for the confounding effects of varying genetic backgrounds, GAA-79 and TTC-79 plasmids were propagated in a set of isogenic strains, M152 (recA) and MM28 (wild-type) (Table 1). Again, GAA-79 was significantly more unstable than TTC-79 in the recA strain (Figure 2B and C), indicating that the orientation-dependent instability is specifically due to the deficiency of RecA. Furthermore, since GAA-79 was significantly more unstable than TTC-79 in the recA strain, and there was no difference in the instability of TTC-79 between the two strains (P = 0.163; Figure 2C), it indicates that the orientation-dependent instability in the absence of RecA is due to an increase in instability when GAA is the lagging strand template.Figure 2.

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