TY - JOUR
T1 - Assembly of the bacteriophage T4 primosome
T2 - Single-molecule and ensembled studies
AU - Zhang, Zhiquan
AU - Spiering, Michelle M.
AU - Trakselis, Michael A.
AU - Ishmael, Faoud T.
AU - Xi, Jun
AU - Benkovic, Stephen J.
AU - Hammes, Gordon G.
PY - 2005/3/1
Y1 - 2005/3/1
N2 - Within replisomes for DNA replication, the primosome is responsible for unwinding double-stranded DNA and synthesizing RNA primers. Assembly of the bacteriophage T4 primosome on individual molecules of ssDNA or forked DNA (fDNA) has been studied by using FRET microscopy. On either DNA substrate, an ordered process of assembly begins with tight 1:1 binding of ssDNA-binding protein (gp32) and helicase-loading protein (gp59) to the DNA. Magnesium adenosine 5′-O-(3-thiotriphosphate) (MgATPγS) mediates the weak binding of helicase (gp41) to DNA coated with gp32 and gp59, whereas MgATP induces gp32 and gp59 to dissociate, leaving gp41 bound to the DNA. Finally, primase (gp61) binds to the gp41·DNA complex. Ensemble studies were used to determine protein stoichiometries and binding constants. These single-molecule studies provide an unambiguous description of the pathway for assembly of the primosome on the lagging strand of DNA at a replication fork.
AB - Within replisomes for DNA replication, the primosome is responsible for unwinding double-stranded DNA and synthesizing RNA primers. Assembly of the bacteriophage T4 primosome on individual molecules of ssDNA or forked DNA (fDNA) has been studied by using FRET microscopy. On either DNA substrate, an ordered process of assembly begins with tight 1:1 binding of ssDNA-binding protein (gp32) and helicase-loading protein (gp59) to the DNA. Magnesium adenosine 5′-O-(3-thiotriphosphate) (MgATPγS) mediates the weak binding of helicase (gp41) to DNA coated with gp32 and gp59, whereas MgATP induces gp32 and gp59 to dissociate, leaving gp41 bound to the DNA. Finally, primase (gp61) binds to the gp41·DNA complex. Ensemble studies were used to determine protein stoichiometries and binding constants. These single-molecule studies provide an unambiguous description of the pathway for assembly of the primosome on the lagging strand of DNA at a replication fork.
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U2 - 10.1073/pnas.0500327102
DO - 10.1073/pnas.0500327102
M3 - Article
C2 - 15728347
AN - SCOPUS:14744278444
SN - 0027-8424
VL - 102
SP - 3254
EP - 3259
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 9
ER -