Repetitive shuttling of a motor protein on DNA.
Many helicases modulate recombination, an essential process that needs to be tightly controlled. Mutations in some human disease helicases cause increased recombination, genome instability and cancer. To elucidate the potential mode of action of these enzymes, here we developed a single-molecule fluorescence assay that can visualize DNA binding and translocation of Escherichia coli Rep, a superfamily 1 DNA helicase homologous to Saccharomyces cerevisiae Srs2. Individual Rep monomers were observed to move on single-stranded (ss)DNA in the 3' to 5' direction using ATP hydrolysis. Strikingly, on hitting a blockade, such as duplex DNA or streptavidin, the protein abruptly snapped back close to its initial position, followed by further cycles of translocation and snapback. This repetitive shuttling is likely to be caused by a blockade-induced protein conformational change that enhances DNA affinity for the protein's secondary DNA binding site, thereby resulting in a transient DNA loop. Repetitive shuttling was also observed on ssDNA bounded by a stalled replication fork and an Okazaki fragment analogue, and the presence of Rep delayed formation of a filament of recombination protein RecA on ssDNA. Thus, the binding of a single Rep monomer to a stalled replication fork can lead to repetitive shuttling along the single-stranded region, possibly keeping the DNA clear of toxic recombination intermediates.
Myong
Sua
S
Physics Department, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, USA.
Rasnik
Ivan
I
Joo
Chirlmin
C
Lohman
Timothy M
TM
Ha
Taekjip
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009068:Movement; D011938:Rec A Recombinases / Q000378:metabolism; D011995:Recombination, Genetic; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D020409:Molecular Motor Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D004265:DNA Helicases