Deficiency of RecA-dependent RecFOR and RecBCD pathways causes increased instability of the (GAA*TTC)n sequence when GAA is the lagging strand template.
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.
Pollard
Laura M
LM
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Chutake
Yogesh K
YK
Rindler
Paul M
PM
Bidichandani
Sanjay I
SI
D001426:Bacterial Proteins / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D043211:Exodeoxyribonuclease V / Q000378:metabolism; D005621:Friedreich Ataxia / Q000235:genetics / Q000378:metabolism; D006801:Humans; D033862:Iron-Binding Proteins / Q000235:genetics; D053842:Microsatellite Instability; D011938:Rec A Recombinases / Q000378:metabolism; D000096923:Frataxin
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D033862:Iron-Binding Proteins; D011938:Rec A Recombinases; D000096923:Frataxin; C081006:RecO protein, E coli; C065831:RecR protein, Bacteria; C066218:recF protein, Bacteria