Abstract
To ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate. After encountering an obstacle, a progressing replisome often aborts DNA synthesis but continues to unwind. However, little is known about how DNA synthesis is resumed downstream of an obstacle. Here, we examine the consequences of a non-replicating replisome collision with a co-directional RNA polymerase (RNAP). Using single-molecule and ensemble methods, we find that T7 helicase interacts strongly with a non-replicating T7 DNA polymerase (DNAP) at a replication fork. As the helicase advances, the associated DNAP also moves forward. The presence of the DNAP increases both helicase’s processivity and unwinding rate. We show that such a DNAP, together with its helicase, is indeed able to actively disrupt a stalled transcription elongation complex, and then initiates replication using the RNA transcript as a primer. These observations exhibit T7 helicase’s novel role in replication re-initiation.
Highlights
To ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate
We recently demonstrated that T7 DNA polymerase (DNAP), working in conjunction with helicase through specific helicase–DNAP interactions, is able to replicate through a leading-strand cyclobutane pyrimidine dimer (CPD) lesion[15] and this has been observed in other systems[25]
During leading-strand replication, DNA synthesis by an actively elongating DNAP has been shown to facilitate T7 helicase unwinding[21, 26, 27]. It is unclear if the non-replicating DNAP, which is disengaged from DNA synthesis, as could occur after a replisome encountering a lesion, still affects helicase unwinding
Summary
To ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate. We recently demonstrated that T7 DNAP, working in conjunction with helicase through specific helicase–DNAP interactions, is able to replicate through a leading-strand cyclobutane pyrimidine dimer (CPD) lesion[15] and this has been observed in other systems[25] Such a direct lesion bypass event occurred in only about 28% of the T7 replisomes, while the remaining population continued helicase unwinding without DNA synthesis beyond the lesion. This suggests the possible existence of other mechanisms for replication re-initiation downstream of the damage. These findings reveal a novel pathway of replication re-initiation enabled by the participation of a replicative helicase
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