Escherichia coli DNA polymerase III holoenzyme footprints three helical turns of its primer.
Escherichia coli DNA polymerase III holoenzyme in the presence of ATP and E. coli single-stranded DNA-binding protein forms an initiation complex on a primed template capable of rapid and highly processive DNA replication. DNase I digestion of initiation complexes demonstrated that holoenzyme protected 27-30 nucleotides of primer. Like the formation of initiation complexes, this protection required both ATP and E. coli single-stranded DNA-binding protein. Initiation complexes assembled with core DNA polymerase III (alpha, epsilon, and theta subunits), gamma-complex (gamma, delta, delta', chi, and omega) and the beta subunit produced a footprint identical to that formed with intact holoenzyme, indicating that initiation complexes formed with reconstituted enzyme and those formed with holoenzyme were equivalent. The presence of the tau subunit in reconstituted initiation complexes did not alter the DNase I footprint. Preinitiation complexes (gamma-complex plus beta subunit) assembled onto primer-template in an ATP-dependent reaction protected a larger region of the primer than did holoenzyme. The addition of core DNA polymerase III to preintiation complexes restored the 30-nucleotide footprint observed with intact holoenzyme. These results suggest that holoenzyme subunits rearrange during initiation complex formation.
Reems
J A
JA
Department of Biochemistry, Biophysics, and Genetics, University of Colorado Health Sciences Center, Denver 80262.
McHenry
C S
CS
D000255:Adenosine Triphosphate / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D009690:Nucleic Acid Conformation
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; D004258:DNA Polymerase III; D003850:Deoxyribonuclease I