Replisome structure suggests mechanism for continuous fork progression and post-replication repair

Abstract

What happens to DNA replication when it encounters a damaged or nicked DNA template has been under investigation for five decades. Initially it was thought that DNA polymerase, and thus the replication-fork progression, would stall at road blocks. After the discovery of replication-fork helicase and replication re-initiation factors by the 1990s, it became clear that the replisome can "skip" impasses and finish replication with single-stranded gaps and double-strand breaks in the product DNA. But the mechanism for continuous fork progression after encountering roadblocks is entangled with translesion synthesis, replication fork reversal and recombination repair. The recently determined structure of the bacteriophage T7 replisome offers the first glimpse of how helicase, primase, leading-and lagging-strand DNA polymerases are organized around a DNA replication fork. The tightly coupled leading-strand polymerase and lagging-strand helicase provides a scaffold to consolidate data accumulated over the past five decades and offers a fresh perspective on how the replisome may skip lesions and complete discontinuous DNA synthesis. Comparison of the independently evolved bacterial and eukaryotic replisomes suggests that repair of discontinuous DNA synthesis occurs post replication in both.

Keywords: Helicase reload; Lesion skipping; Polymerase restart; Replication fork; Replisome.

Fig. 1.

Diagrams of DNA replisomes. (A) Low-resolution cryoEM reconstitution of yeast CMG, Pol ε and primosome complex reveals a three-tier-core structure. Diagrams of the DNA fork substrate in the three-tier-core structure. The conserved DNA part are shown in orange, and the different downstream parental DNA in bacterial and eukaryotic replisome is shown in gold and silver, respectively. (B) A hand-over-hand mechanism of replication-fork helicase translocation along DNA. (C) A textbook diagram of the replisome and directions of the three motor proteins along the DNA fork substrate. (D) Structure of the T7 replisome. The “T” shaped DNA fork is highlighted by a semi-transparent T.

Fig. 2.

Mechanisms for lesion skipping by bacterial replisome along the leading strand. (A) DNA replicative polymerase bypasses a minor DNA base lesion, e.g. 8-oxoG. (B) Lesion bypass by TLS polymerase, which requires at least two polymerase-switch steps before the polymerase and helicase re-association. (C) When a leading-strand polymerase is completely stalled by a roadblock, e.g. UV-induced pyrimidine dimers, the leading strand DNA synthesis requires a new primer to restart. The incomplete DNA synthesis due to the lesion may be completed by TLS or template-switching synthesis (TSS) post replication.

Fig. 3.

Mechanisms for lesion skipping by bacterial replisome along the lagging strand. (A) Most lagging-strand lesions have no ill effect on the helicase and are skipped by primase and polymerase alike. (B) A nick in the lagging strand leads to helicase dissociation, while the leading strand polymerase continues at a reduced speed. (C) PriA may displace the leading strand DNA polymerase, and together with PriB, PriC and DnaT help to reload DnaB. The double-strand break in the lagging strand depends on homologous recombination for repair.

Fig. 4.

Mechanisms for lesion skipping by eukaryotic replisome. (A) Base lesions in either leading or lagging strands. When a leading-strand polymerase is completely stalled, to continue DNA synthesis requires a new primer to restart and reloading of Pol ε. The gapped DNA synthesis product due to the lesion may be repaired by TLS or template-switching synthesis post replication. (B) Nicks in either template strand lead to double strand breaks after replication. If the leading strand is nicked, replication can be completed only by a fork approaching from the other side as CMG cannot be reloaded. The resulting double-strand breaks in either leading or lagging strands are repaired by homologous recombination (HR). (C) In the presence of an ICL, the majority of replisomes are able to traverse the lesion and continue DNA synthesis on the other side of the ICL. The lesion, gap and break in the replication product are removed by repair pathways including DNA incision, TLS and HR.