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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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(GAA·TTC)n constructs used to analyze repeat instability. (GAA·TTC)n sequences of the indicated lengths were cloned into the Pst I/Xba I sites of pUC19 in both orientations relative to the unidirectional pMB1 origin of replication. Repeat-containing plasmids are depicted in either the ‘GAA’ or ‘TTC’ orientations, based on whether (GAA)n or (TTC)n serves as the lagging strand template, respectively. The plasmid constructs contain repeat lengths of n = 21, 41 and 79. The black boxes flanking the repeat represent minimal flanking sequence from intron 1 of the FXN gene.
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Figure 1: (GAA·TTC)n constructs used to analyze repeat instability. (GAA·TTC)n sequences of the indicated lengths were cloned into the Pst I/Xba I sites of pUC19 in both orientations relative to the unidirectional pMB1 origin of replication. Repeat-containing plasmids are depicted in either the ‘GAA’ or ‘TTC’ orientations, based on whether (GAA)n or (TTC)n serves as the lagging strand template, respectively. The plasmid constructs contain repeat lengths of n = 21, 41 and 79. The black boxes flanking the repeat represent minimal flanking sequence from intron 1 of the FXN gene.

Mentions: (GAA·TTC)n repeats with minimal flanking intron 1 sequence were cloned in the Pst I/Xba I sites of pUC19 using PCR products of the FXN gene from human subjects as previously described (25). The following recombinant plasmids, with the repeat tract cloned in both orientations with respect to the pMB1 origin of replication, were confirmed by sequencing and selected for further analysis (sequences in the TTC and GAA orientations were identical and only the sequence of the ‘GAA’ orientation is shown here): GAA-21 [(GAA)17(A)(GAA)4], GAA-41 [(GAA)37(A)(GAA)4], and GAA-79 [(GAA)79] (Figure 1). Deletion of the Plac promoter in pUC19, to produce the pDEL-GAA-79 construct (Figure 3A), was accomplished by first introducing an Apa I site at the −35 position using the QuikChange II XL site-directed mutagenesis kit (Stratagene), followed by removal of the fragment between Apa I and Hind III, thus deleting both the −10 and −35 sites. The lack of transcription was confirmed via loss of ability to produce blue colonies on X-gal containing plates. The (GAA)79 insert from the original pUC19 construct was subcloned in the Pst I/Xba I sites to produce pDEL-GAA-79 and confirmed by sequencing. Another plasmid (pINS-GAA-79) (Figure 3A) was created to alter the distance between the origin of replication and the GAA triplet-repeat by inserting a 1525 bp sequence from intron 1 of the human FXN gene. The following primers were used to amplify the spacer sequence from human genomic DNA:Figure 1.


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)

(GAA·TTC)n constructs used to analyze repeat instability. (GAA·TTC)n sequences of the indicated lengths were cloned into the Pst I/Xba I sites of pUC19 in both orientations relative to the unidirectional pMB1 origin of replication. Repeat-containing plasmids are depicted in either the ‘GAA’ or ‘TTC’ orientations, based on whether (GAA)n or (TTC)n serves as the lagging strand template, respectively. The plasmid constructs contain repeat lengths of n = 21, 41 and 79. The black boxes flanking the repeat represent minimal flanking sequence from intron 1 of the FXN gene.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: (GAA·TTC)n constructs used to analyze repeat instability. (GAA·TTC)n sequences of the indicated lengths were cloned into the Pst I/Xba I sites of pUC19 in both orientations relative to the unidirectional pMB1 origin of replication. Repeat-containing plasmids are depicted in either the ‘GAA’ or ‘TTC’ orientations, based on whether (GAA)n or (TTC)n serves as the lagging strand template, respectively. The plasmid constructs contain repeat lengths of n = 21, 41 and 79. The black boxes flanking the repeat represent minimal flanking sequence from intron 1 of the FXN gene.
Mentions: (GAA·TTC)n repeats with minimal flanking intron 1 sequence were cloned in the Pst I/Xba I sites of pUC19 using PCR products of the FXN gene from human subjects as previously described (25). The following recombinant plasmids, with the repeat tract cloned in both orientations with respect to the pMB1 origin of replication, were confirmed by sequencing and selected for further analysis (sequences in the TTC and GAA orientations were identical and only the sequence of the ‘GAA’ orientation is shown here): GAA-21 [(GAA)17(A)(GAA)4], GAA-41 [(GAA)37(A)(GAA)4], and GAA-79 [(GAA)79] (Figure 1). Deletion of the Plac promoter in pUC19, to produce the pDEL-GAA-79 construct (Figure 3A), was accomplished by first introducing an Apa I site at the −35 position using the QuikChange II XL site-directed mutagenesis kit (Stratagene), followed by removal of the fragment between Apa I and Hind III, thus deleting both the −10 and −35 sites. The lack of transcription was confirmed via loss of ability to produce blue colonies on X-gal containing plates. The (GAA)79 insert from the original pUC19 construct was subcloned in the Pst I/Xba I sites to produce pDEL-GAA-79 and confirmed by sequencing. Another plasmid (pINS-GAA-79) (Figure 3A) was created to alter the distance between the origin of replication and the GAA triplet-repeat by inserting a 1525 bp sequence from intron 1 of the human FXN gene. The following primers were used to amplify the spacer sequence from human genomic DNA:Figure 1.

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