Mapping fast DNA polymerase exchange during replication

Abstract

AbstractDespite extensive studies on DNA replication, the exchange mechanisms of DNA polymerase during replication remain unclear. Existing models propose that this exchange is facilitated by protein partners like helicase. Here we present data, employing a combination of mechanical DNA manipulation and single fluorescent protein observation, that reveal DNA polymerase undergoing rapid and autonomous exchange during replication not coordinated by other proteins. The DNA polymerase shows fast unbinding and rebinding dynamics, displaying a preference for either exonuclease or polymerase activity, or pausing events, during each brief binding event. We also observed a ‘memory effect’ in DNA polymerase rebinding, i.e., the enzyme tends to preserve its prior activity upon reassociation. This effect, potentially linked to the ssDNA/dsDNA junction’s conformation, might play a role in regulating binding preference enabling high processivity amidst rapid protein exchange. Taken together, our findings support an autonomous replication model that includes rapid protein exchange, burst of activity, and a ‘memory effect’ while moving processively forward.