期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 102, 期 9, 页码 3254-3259出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0500327102
关键词
DNA replication; fluorescence microscopy; fluorescence-resonance energy transfer
资金
- NIGMS NIH HHS [R01 GM065128, GM13306, GM65128, GM071130, R01 GM013306, F32 GM071130] Funding Source: Medline
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 (f DNA) 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 9p41 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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