TY - JOUR
T1 - DNA-induced dimerization of the Escherichia coli Rep helicase
AU - Chao, Kinlin
AU - Lohman, Timothy M.
N1 - Funding Information:
PVr thank David Mascotti for determing the Rep monomer s&r size on poly(&A). Drs W. Bujalowski and I. Wong for valuable discussions. Mike Green and Bill Van Zante for help with oligodeoxynucleotide purification and Lisa Lohman for help with preparing the figures. This research was supported in part by grants from the Sational 1nst)itutes of Health (GM30498). the American (lancer Society (NP-756). the Robert A. Welch Foundation (&i-898) and the Texas Agricult,ural Experiment Station. T.M.L. is the recipient of American (‘anerr Society Facault,y Researrh Award (FRA-303).
PY - 1991/10/20
Y1 - 1991/10/20
N2 - The Escherichia coli Rep protein is a DNA helicase that is involved in DNA replication. We have examined the effects of DNA binding on the assembly state of the Rep protein using small-zone gel permeation chromatography and chemical crosslinking of the protein. Complexes of Rep protein were formed with short single-stranded and duplex hairpin oligodeoxynucleotides with lengths such that only a single Rep monomer could bind per oligodeoxynucleotide (i.e. 2 Rep monomers could not bind contiguously on the oligodeoxynucleotides). In the absence of DNA, Rep protein is monomeric (Mr 72,800) up to concentrations of at least 8 μm (monomer), even in the presence of its nucleotide cofactors (ATP, ADP, ATP-γ-S). However, the binding of Rep monomers to single-stranded (ss) oligodeoxynucleotides, d(pN)n (12 ≤ n ≤ 20), induces the Rep monomers to oligomerize. Upon treatment of the Rep-ss oligodeoxynucleotide complexes with the protein crosslinking reagent dimethyl-suberimidate (DMS) and subsequent removal of the DNA, crosslinked Rep dimers are observed, independent of oligodeoxynucleotide length (n ≤ 20). Furthermore, short duplex oligodeoxynucleotides also induce the Rep monomers to dimerize. Formation of the Rep dimers results from an actual DNA-induced dimerization, rather than the adventitious crosslinking of Rep monomers bound contiguously to a single oligodeoxynucleotide. The purified DMS-crosslinked Rep dimer shows increased affinity for DNA and retains DNA-dependent ATPase and DNA helicase activities, as shown by its ability to unwind M13 RF DNA in the presence of the bacteriophage f1 gene II protein. On the basis of these observations and since the dimer is the major species when Rep is bound to DNA, we suggest that a DNA-induced Rep dimer is the functionally active form of the Rep helicase.
AB - The Escherichia coli Rep protein is a DNA helicase that is involved in DNA replication. We have examined the effects of DNA binding on the assembly state of the Rep protein using small-zone gel permeation chromatography and chemical crosslinking of the protein. Complexes of Rep protein were formed with short single-stranded and duplex hairpin oligodeoxynucleotides with lengths such that only a single Rep monomer could bind per oligodeoxynucleotide (i.e. 2 Rep monomers could not bind contiguously on the oligodeoxynucleotides). In the absence of DNA, Rep protein is monomeric (Mr 72,800) up to concentrations of at least 8 μm (monomer), even in the presence of its nucleotide cofactors (ATP, ADP, ATP-γ-S). However, the binding of Rep monomers to single-stranded (ss) oligodeoxynucleotides, d(pN)n (12 ≤ n ≤ 20), induces the Rep monomers to oligomerize. Upon treatment of the Rep-ss oligodeoxynucleotide complexes with the protein crosslinking reagent dimethyl-suberimidate (DMS) and subsequent removal of the DNA, crosslinked Rep dimers are observed, independent of oligodeoxynucleotide length (n ≤ 20). Furthermore, short duplex oligodeoxynucleotides also induce the Rep monomers to dimerize. Formation of the Rep dimers results from an actual DNA-induced dimerization, rather than the adventitious crosslinking of Rep monomers bound contiguously to a single oligodeoxynucleotide. The purified DMS-crosslinked Rep dimer shows increased affinity for DNA and retains DNA-dependent ATPase and DNA helicase activities, as shown by its ability to unwind M13 RF DNA in the presence of the bacteriophage f1 gene II protein. On the basis of these observations and since the dimer is the major species when Rep is bound to DNA, we suggest that a DNA-induced Rep dimer is the functionally active form of the Rep helicase.
KW - ATPase
KW - helicase
KW - protein-DNA interactions
KW - replication
UR - http://www.scopus.com/inward/record.url?scp=0026054936&partnerID=8YFLogxK
U2 - 10.1016/0022-2836(91)90926-W
DO - 10.1016/0022-2836(91)90926-W
M3 - Article
C2 - 1658335
AN - SCOPUS:0026054936
VL - 221
SP - 1165
EP - 1181
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 4
ER -