Zhang Zhiquan, Spiering Michelle M, Trakselis Michael A, Ishmael Faoud T, Xi Jun, Benkovic Stephen J, Hammes Gordon G
Department of Biochemistry, Duke University Medical Center, Box 3711, Durham, NC 27710, USA.
Proc Natl Acad Sci U S A. 2005 Mar 1;102(9):3254-9. doi: 10.1073/pnas.0500327102. Epub 2005 Feb 22.
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) (MgATPgammaS) 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.
在用于DNA复制的复制体中，引发体负责解开双链DNA并合成RNA引物。通过荧光共振能量转移显微镜（FRET显微镜）研究了噬菌体T4引发体在单链DNA（ssDNA）或叉状DNA（fDNA）单个分子上的组装。在任何一种DNA底物上，组装的有序过程始于单链DNA结合蛋白（gp32）和解旋酶加载蛋白（gp59）与DNA的紧密1:1结合。镁腺苷5'-O-（3-硫代三磷酸）（MgATPγS）介导解旋酶（gp41）与包被有gp32和gp59的DNA的弱结合，而MgATP诱导gp32和gp59解离，使gp41与DNA结合。最后，引发酶（gp61）与gp41-DNA复合物结合。整体研究用于确定蛋白质化学计量和结合常数。这些单分子研究明确描述了引发体在复制叉处DNA后随链上的组装途径。
