Mycobacterium tuberculosis RecG protein but not RuvAB or RecA protein is efficient at remodeling the stalled replication forks: implications for multiple mechanisms of replication restart in mycobacteria.
Aberrant DNA replication, defects in the protection, and restart of stalled replication forks are major causes of genome instability in all organisms. Replication fork reversal is emerging as an evolutionarily conserved physiological response for restart of stalled forks. Escherichia coli RecG, RuvAB, and RecA proteins have been shown to reverse the model replication fork structures in vitro. However, the pathways and the mechanisms by which Mycobacterium tuberculosis, a slow growing human pathogen, responds to different types of replication stress and DNA damage are unclear. Here, we show that M. tuberculosis RecG rescues E. coli ΔrecG cells from replicative stress. The purified M. tuberculosis RecG (MtRecG) and RuvAB (MtRuvAB) proteins catalyze fork reversal of model replication fork structures with and without a leading strand single-stranded DNA gap. Interestingly, single-stranded DNA-binding protein suppresses the MtRecG- and MtRuvAB-mediated fork reversal with substrates that contain lagging strand gap. Notably, our comparative studies with fork structures containing template damage and template switching mechanism of lesion bypass reveal that MtRecG but not MtRuvAB or MtRecA is proficient in driving the fork reversal. Finally, unlike MtRuvAB, we find that MtRecG drives efficient reversal of forks when fork structures are tightly bound by protein. These results provide direct evidence and valuable insights into the underlying mechanism of MtRecG-catalyzed replication fork remodeling and restart pathways in vivo.
From the Department of Biochemistry, Indian Institute of Science, Bangalore-560012, India.
Roshan Singh
RS
Thakur
From the Department of Biochemistry, Indian Institute of Science, Bangalore-560012, India.
Shivakumar
S
Basavaraju
From the Department of Biochemistry, Indian Institute of Science, Bangalore-560012, India.
Jasbeer Singh
JS
Khanduja
From the Department of Biochemistry, Indian Institute of Science, Bangalore-560012, India.
K
K
Muniyappa
From the Department of Biochemistry, Indian Institute of Science, Bangalore-560012, India nganesh@biochem.iisc.ernet.in.
Ganesh
G
Nagaraju
D001426:Bacterial Proteins / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D016680:Genome, Bacterial; D042822:Genomic Instability; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides; D011938:Rec A Recombinases / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D011938:Rec A Recombinases; D004265:DNA Helicases
DNA damage; DNA helicase; DNA replication; Mycobacterium tuberculosis; genomic instability