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Termination of replication occurs when two forks converge, an important but understudied process. In PNAS, a report from the Courcelle group examines replication termination using deep-sequencing genomic profiling of replicating cells to obtain copy number information about head-on collision of replication forks in different genetic backgrounds (1). Mutations in the SbcC-SbcD (SbcCD) and ExoI nucleases of Escherichia coli result in overreplication of DNA at the terminal replication zone where forks converge, implying that extra DNA is made upon termination and these nucleases are needed to excise the extra DNA. Furthermore, mutational studies of the RecBCD helicase/nuclease reveal that it acts at a step after SbcCD/Exo1 action to complete the processing of overreplicated DNA generated by fork convergence. Overreplication upon termination in E. coli has been reported earlier, but the DNA structures produced, and subsequent processing steps are not well understood (2⇓–4). The report by Wendel et al. (1) demonstrates that termination of replication is a complex process orchestrated by many factors, and implies specific roles of the enzymes involved. Termination of replication, when two replication forks meet head-on, has the potential for deleterious consequences. For example, amplifications, resections leading to deletions, and other DNA rearrangements are associated with defective replication termination (1⇓⇓–4). Extensive studies have outlined the events that activate origins and advance replication forks in bacteria and eukaryotes (5, 6), but little is known about the replication termination process, possibly because termination does not occur at a defined sequence, making it difficult to study. The circular chromosome of E. coli has been an attractive model to study the termination process for two main reasons. First, E. coli has only one origin ( oriC ) that forms bidirectional forks that meet head-on roughly half way around the circular genome from the origin. Second, E. … [↵][1]1To whom correspondence should be addressed. Email: odonnel{at}rockefeller.edu. [1]: #xref-corresp-1-1

DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

The concentration of ribonucleoside triphosphates (rNTPs) in cells is far greater than the concentration of deoxyribonucleoside triphosphates (dNTPs), and this pool imbalance presents a challenge for DNA polymerases (Pols) to select their proper substrate. This report examines the effect of nucleotide pool imbalance on the rate and fidelity of the Escherichia coli replisome. We find that rNTPs decrease replication fork rate by competing with dNTPs at the active site of the C-family Pol III replicase at a step that does not require correct base-pairing. The effect of rNTPs on Pol rate generalizes to B-family eukaryotic replicases, Pols δ and ε. Imbalance of the dNTP pool also slows the replisome and thus is not specific to rNTPs. We observe a measurable frequency of rNMP incorporation that predicts one rNTP incorporated every 2.3 kb during chromosome replication. Given the frequency of rNMP incorporation, the repair of rNMPs is likely rapid. RNase HII nicks DNA at single rNMP residues to initiate replacement with dNMP. Considering that rNMPs will mark the new strand, RNase HII may direct strand-specificity for mismatch repair (MMR). How the newly synthesized strand is recognized for MMR is uncertain in eukaryotes and most bacteria, which lack a methyl-directed nicking system. Here we demonstrate that Bacillus subtilis incorporates rNMPs in vivo, that RNase HII plays a role in their removal, and the RNase HII gene deletion enhances mutagenesis, suggesting a possible role of incorporated rNMPs in MMR.