TY - JOUR
T1 - E. coli Rep oligomers are required to initiate DNA unwinding in vitro
AU - Cheng, Wei
AU - Hsieh, John
AU - Brendza, Katherine M.
AU - Lohman, Timothy M.
N1 - Funding Information:
We thank members of the laboratory, especially Janid Ali, Karl Maluf, Aaron Lucius, Chris Fischer and David Levine for their critical comments on the manuscript and input during the course of these experiments, John Majors, Taekjip Ha and Wlodek Bujalowski for critical comments on the manuscript, and Thang Ho for synthesis and purification of the oligodeoxynucleotides. This work was supported, in part, by a grant from the NIH (GM 45948) and by a William M. Keck Foundation postdoctoral fellowship to K.M.B.
PY - 2001/7/6
Y1 - 2001/7/6
N2 - E. coli Rep protein is a 3′ to 5′ SF1 superfamily DNA helicase which is monomeric in the absence of DNA, but can dimerize upon binding either single-stranded or duplex DNA. A variety of biochemical studies have led to proposals that Rep dimerization is important for its helicase activity; however, recent structural studies of Bacillus stearothermophilus PcrA have led to suggestions that SF1 helicases, such as E. coli Rep and E. coli UvrD, function as monomeric helicases. We have examined the question of whether Rep oligomerization is important for its DNA helicase activity using pre-steady state stopped-flow and chemical quenched-flow kinetic studies of Rep-catalyzed DNA unwinding. The results from four independent experiments demonstrate that Rep oligomerization is required for initiation of DNA helicase activity in vitro. No DNA unwinding is observed when only a Rep monomer is bound to the DNA substrate, even when fluorescent DNA substrates are used that can detect partial unwinding of the first few base-pairs at the ss-ds-DNA junction. In fact, under these conditions, ATP hydrolysis causes dissociation of the Rep monomer from the DNA, rather than DNA unwinding. These studies demonstrate that wild-type Rep monomers are unable to initiate DNA unwinding in vitro, and that oligomerization is required.
AB - E. coli Rep protein is a 3′ to 5′ SF1 superfamily DNA helicase which is monomeric in the absence of DNA, but can dimerize upon binding either single-stranded or duplex DNA. A variety of biochemical studies have led to proposals that Rep dimerization is important for its helicase activity; however, recent structural studies of Bacillus stearothermophilus PcrA have led to suggestions that SF1 helicases, such as E. coli Rep and E. coli UvrD, function as monomeric helicases. We have examined the question of whether Rep oligomerization is important for its DNA helicase activity using pre-steady state stopped-flow and chemical quenched-flow kinetic studies of Rep-catalyzed DNA unwinding. The results from four independent experiments demonstrate that Rep oligomerization is required for initiation of DNA helicase activity in vitro. No DNA unwinding is observed when only a Rep monomer is bound to the DNA substrate, even when fluorescent DNA substrates are used that can detect partial unwinding of the first few base-pairs at the ss-ds-DNA junction. In fact, under these conditions, ATP hydrolysis causes dissociation of the Rep monomer from the DNA, rather than DNA unwinding. These studies demonstrate that wild-type Rep monomers are unable to initiate DNA unwinding in vitro, and that oligomerization is required.
KW - FRET
KW - Helicase
KW - Motors
KW - Oligomerization
KW - Pre-steady state kinetics
UR - http://www.scopus.com/inward/record.url?scp=0035816217&partnerID=8YFLogxK
U2 - 10.1006/jmbi.2001.4758
DO - 10.1006/jmbi.2001.4758
M3 - Article
C2 - 11428893
AN - SCOPUS:0035816217
SN - 0022-2836
VL - 310
SP - 327
EP - 350
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -