Functional Uncoupling of Twin Polymerases MECHANISM OF POLYMERASE DISSOCIATION FROM A LAGGING-STRAND BLOCK*

Replication forks are constantly subjected to events that lead to fork stalling, stopping, or collapse. Using a synthetic rolling circle DNA substrate, we demonstrate that a block to the lagging-strand polymerase does not compromise helicase or leading-strand polymerase activity. In fact, lagging-strand synthesis also continues. Thus, the blocked lagging-strand enzyme quickly dissociates from the block site and resumes synthesis on new primed sites. Furthermore, studies in which the lagging polymerase is continuously blocked show that the leading polymerase continues unabated even as it remains attached to the lagging-strand enzyme. Hence, upon encounter of a block to the lagging stand, the polymerases functionally uncouple yet remain physically associated. Further study reveals that naked single-stranded DNA results in disruption of a stalled polymerase from its (cid:1) -DNA substrate. Thus, as the replisome advances, the single-stranded DNA loop that accumulates on the lag-ging-strand template releases the stalled lagging-strand polymerase from (cid:1) after SSB protein is depleted. The lagging-strand polymerase is then free to continue Okazaki fragment production. ,