The 2B domain of the Escherichia coli Rep protein is not required for DNA helicase activity.
The Escherichia coli Rep protein is a 3' to 5' SF1 DNA helicase required for replication of bacteriophage phiX174 in E. coli, and is structurally homologous to the E. coli UvrD helicase and the Bacillus stearothermophilus PcrA helicase. Previous crystallographic studies of Rep protein bound to single-stranded DNA revealed that it can undergo a large conformational change consisting of an approximately 130 degrees rotation of its 2B subdomain about a hinge region connected to the 2A subdomain. Based on crystallographic studies of PcrA, its 2B subdomain has been proposed to form part of its duplex DNA binding site and to play a role in duplex destabilization. To test the role of the 2B subdomain in Rep-catalyzed duplex DNA unwinding, we have deleted its 2B subdomain, replacing it with three glycines, to form the RepDelta2B protein. This RepDelta2B protein can support phiX174 replication in a rep(-) E. coli strain, although the growth rate of E. coli containing the repDelta2B gene is approximately 1.5-fold slower than with the wild-type rep gene. Pre-steady-state, single-turnover DNA unwinding kinetics experiments show that purified RepDelta2B protein has DNA helicase activity in vitro and unwinds an 18-bp DNA duplex with rates at least as fast as wild-type Rep, and with higher extents of unwinding and higher affinity for the DNA substrate. These studies show that the 2B domain of Rep is not required for DNA helicase activity in vivo or in vitro, and that it does not facilitate DNA unwinding in vitro.
Cheng
Wei
W
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
Brendza
Katherine M
KM
Gauss
George H
GH
Korolev
Sergey
S
Waksman
Gabriel
G
Lohman
Timothy M
TM
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000502:physiology; D010584:Bacteriophage phi X 174 / Q000235:genetics; D004265:DNA Helicases / Q000737:chemistry* / Q000502:physiology; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013379:Substrate Specificity
D004269:DNA, Bacterial; D004279:DNA, Viral; D029968:Escherichia coli Proteins; C506744:rep protein, E coli; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases