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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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Pathways involved in maintaining stability of the (GAA·TTC)n sequence in E. coli (see Discussion section for details). (A) Replication of the (GAA·TTC)n sequence in the GAA orientation may be hypothetically visualized as a ‘lesion’ on the lagging strand. (B) A lagging strand lesion may be bypassed by dissociation of the lagging strand polymerase thereby creating a daughter-strand gap. Gap repair by the RecFOR pathway would ensue, where the RecFOR complex loads RecA on the gapped DNA, thus effecting DNA strand exchange for homologous recombinational repair. (C) Stalling may result in fork regression with formation of a Holliday junction. The Holliday junction may be resolved without cleavage via exonucleolytic digestion by the helicase-nuclease combination of RuvAB and RecBCD, so that a fork structure is restored. (D) Stalled forks may break, and repair of such a break with restart of replication may be achieved by joint molecule formation between the intact and broken sister arms via the combined action of RecBCD, RuvAB and RecA.
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Figure 8: Pathways involved in maintaining stability of the (GAA·TTC)n sequence in E. coli (see Discussion section for details). (A) Replication of the (GAA·TTC)n sequence in the GAA orientation may be hypothetically visualized as a ‘lesion’ on the lagging strand. (B) A lagging strand lesion may be bypassed by dissociation of the lagging strand polymerase thereby creating a daughter-strand gap. Gap repair by the RecFOR pathway would ensue, where the RecFOR complex loads RecA on the gapped DNA, thus effecting DNA strand exchange for homologous recombinational repair. (C) Stalling may result in fork regression with formation of a Holliday junction. The Holliday junction may be resolved without cleavage via exonucleolytic digestion by the helicase-nuclease combination of RuvAB and RecBCD, so that a fork structure is restored. (D) Stalled forks may break, and repair of such a break with restart of replication may be achieved by joint molecule formation between the intact and broken sister arms via the combined action of RecBCD, RuvAB and RecA.

Mentions: Hypothetically visualizing replication through the (GAA·TTC)n sequence in the GAA orientation as a ‘lesion’ on the lagging strand (Figure 8A), we propose three non-mutually exclusive mechanisms by which repeat stability would be maintained (Figure 8B–D). A lagging strand lesion may be bypassed by dissociation of the lagging strand polymerase from the template and restarting replication of the nascent lagging strand on the 3′ side of the lesion thereby creating a daughter-strand gap (Figure 8B). Such a gap would be repaired by the RecFOR pathway, where the RecFOR complex loads RecA on to the gapped DNA that is precoated with SSB, thus effecting DNA strand exchange for homologous recombinational repair (58). Consistent with the importance of this pathway in maintaining repeat stability, both recF and recO mutants resulted in enhanced instability in the GAA orientation. On the other hand, the stalled replication fork may regress wherein the nascent strands pair to form a Holliday junction (Figure 8C). Holliday junctions may be resolved without the need for cleavage by processing of the free double-stranded DNA end via exonucleolytic digestion by the helicase-nuclease combination of RuvAB and RecBCD, so that a fork structure is restored (46,59). However, stalled forks may break (Figure 8D), either spontaneously or by the Holliday junction resolvase RuvC (45,59). Repair of such a break and restart of replication would be achieved by joint molecule formation between the intact and broken sister arms via the combined action of RecBCD and RecA (45). The enhanced instability in the GAA orientation seen in the recBC mutant supports these mechanisms for maintaining repeat stability. The helicase–endonuclease complex RuvABC is involved in both the RecFOR and RecBCD pathways thus explaining the orientation-dependent instability in the ruvAB mutant.Figure 8.


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)

Pathways involved in maintaining stability of the (GAA·TTC)n sequence in E. coli (see Discussion section for details). (A) Replication of the (GAA·TTC)n sequence in the GAA orientation may be hypothetically visualized as a ‘lesion’ on the lagging strand. (B) A lagging strand lesion may be bypassed by dissociation of the lagging strand polymerase thereby creating a daughter-strand gap. Gap repair by the RecFOR pathway would ensue, where the RecFOR complex loads RecA on the gapped DNA, thus effecting DNA strand exchange for homologous recombinational repair. (C) Stalling may result in fork regression with formation of a Holliday junction. The Holliday junction may be resolved without cleavage via exonucleolytic digestion by the helicase-nuclease combination of RuvAB and RecBCD, so that a fork structure is restored. (D) Stalled forks may break, and repair of such a break with restart of replication may be achieved by joint molecule formation between the intact and broken sister arms via the combined action of RecBCD, RuvAB and RecA.
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Related In: Results  -  Collection

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Show All Figures
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Figure 8: Pathways involved in maintaining stability of the (GAA·TTC)n sequence in E. coli (see Discussion section for details). (A) Replication of the (GAA·TTC)n sequence in the GAA orientation may be hypothetically visualized as a ‘lesion’ on the lagging strand. (B) A lagging strand lesion may be bypassed by dissociation of the lagging strand polymerase thereby creating a daughter-strand gap. Gap repair by the RecFOR pathway would ensue, where the RecFOR complex loads RecA on the gapped DNA, thus effecting DNA strand exchange for homologous recombinational repair. (C) Stalling may result in fork regression with formation of a Holliday junction. The Holliday junction may be resolved without cleavage via exonucleolytic digestion by the helicase-nuclease combination of RuvAB and RecBCD, so that a fork structure is restored. (D) Stalled forks may break, and repair of such a break with restart of replication may be achieved by joint molecule formation between the intact and broken sister arms via the combined action of RecBCD, RuvAB and RecA.
Mentions: Hypothetically visualizing replication through the (GAA·TTC)n sequence in the GAA orientation as a ‘lesion’ on the lagging strand (Figure 8A), we propose three non-mutually exclusive mechanisms by which repeat stability would be maintained (Figure 8B–D). A lagging strand lesion may be bypassed by dissociation of the lagging strand polymerase from the template and restarting replication of the nascent lagging strand on the 3′ side of the lesion thereby creating a daughter-strand gap (Figure 8B). Such a gap would be repaired by the RecFOR pathway, where the RecFOR complex loads RecA on to the gapped DNA that is precoated with SSB, thus effecting DNA strand exchange for homologous recombinational repair (58). Consistent with the importance of this pathway in maintaining repeat stability, both recF and recO mutants resulted in enhanced instability in the GAA orientation. On the other hand, the stalled replication fork may regress wherein the nascent strands pair to form a Holliday junction (Figure 8C). Holliday junctions may be resolved without the need for cleavage by processing of the free double-stranded DNA end via exonucleolytic digestion by the helicase-nuclease combination of RuvAB and RecBCD, so that a fork structure is restored (46,59). However, stalled forks may break (Figure 8D), either spontaneously or by the Holliday junction resolvase RuvC (45,59). Repair of such a break and restart of replication would be achieved by joint molecule formation between the intact and broken sister arms via the combined action of RecBCD and RecA (45). The enhanced instability in the GAA orientation seen in the recBC mutant supports these mechanisms for maintaining repeat stability. The helicase–endonuclease complex RuvABC is involved in both the RecFOR and RecBCD pathways thus explaining the orientation-dependent instability in the ruvAB mutant.Figure 8.

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