DYNAMICS OF DNA-TRACKING BY 2 SLIDING-CLAMP PROTEINS

Bacteriophage T4 gene 45 protein (gp45) and Escherichia coli beta are DNA-tracking sliding-clamp proteins that increase processivity by teth ering their conjugate DNA polymerases to DNA, gp45 also activates T4 l ate transcription, DNA loading of gp45 and beta requires ATP or dATP h ydrolysis; efficient loading at peimer-template junctions is assisted by single-stranded DNA-binding proteins, The kinetics of gp45 loading and tracking have been examined by DNase I footprinting of linear DNA with one blunt end, one primer-template junction, and binding sites fo r proteins that block gp45 tracking, DNA loading of gp45 fall also be interrupted by adding the non-hydrolyzable ATP analog ATP-gamma-S, At: saturation, DNA is very closely packed with gp45 or beta. When gp45 l oading is interrupted, or when a segment of the track is blocked off, the gp45 footprint dissipates within seconds, but the DNA-tracking sta te of beta is much more stable, The stability of the tracking state of gp45 is, however, increased by the macromolecular crowding agent poly ethylene glycol, We suggest that labile gp45 catenation directly gener ates the coupling of late transcription to DIVA replication during bac teriophage T4 multiplication.