Nina Y. Yao

Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork

Eukaryotes use distinct polymerases for leading- and lagging-strand replication, but how they target their respective strands is uncertain. We reconstituted Saccharomyces cerevisiae replication forks and found that CMG helicase selects polymerase (Pol) ɛ to the exclusion of Pol δ on the leading strand. Even if Pol δ assembles on the leading strand, Pol ɛ rapidly replaces it. Pol δ–PCNA is distributive with CMG, in contrast to its high stability on primed ssDNA. Hence CMG will not stabilize Pol δ, instead leaving the leading strand accessible for Pol ɛ and stabilizing Pol ɛ. Comparison of Pol ɛ and Pol δ on a lagging-strand model DNA reveals the opposite. Pol δ dominates over excess Pol ɛ on PCNA-primed ssDNA. Thus, PCNA strongly favors Pol δ over Pol ɛ on the lagging strand, but CMG over-rides and flips this balance in favor of Pol ɛ on the leading strand.

CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication

Significance All cells must replicate their chromosomes prior to cell division. This process is carried out by a collection of proteins, known as the replisome, that act together to unwind the double helix and synthesize two new DNA strands complementary to the two parental strands. The details of replisome function have been worked out for bacteria but are much less well understood for eukaryotic cells. We have developed a system for studying eukaryotic replisome function in vitro using purified proteins. Using this system, we have identified a direct interaction between the component that unwinds the DNA, the CMG (Cdc45-MCM-GINS) helicase, and the component that replicates the leading strand, DNA polymerase ε, to form a large helicase–polymerase holoenzyme comprising 15 separate proteins. 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.

Single-molecule analysis reveals that the lagging strand increases replisome processivity but slows replication fork progression

Single-molecule techniques are developed to examine mechanistic features of individual E. coli replisomes during synthesis of long DNA molecules. We find that single replisomes exhibit constant rates of fork movement, but the rates of different replisomes vary over a surprisingly wide range. Interestingly, lagging strand synthesis decreases the rate of the leading strand, suggesting that lagging strand operations exert a drag on replication fork progression. The opposite is true for processivity. The lagging strand significantly increases the processivity of the replisome, possibly reflecting the increased grip to DNA provided by 2 DNA polymerases anchored to sliding clamps on both the leading and lagging strands.

Mechanism of Proliferating Cell Nuclear Antigen Clamp Opening by Replication Factor C

The eukaryotic replication factor C (RFC) clamp loader is an AAA+ spiral-shaped heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) clamp processivity factor on DNA. In this study, we examined the roles of individual RFC subunits in opening the PCNA clamp. Interestingly, Rfc1, which occupies the position analogous to the δ clamp-opening subunit in the Escherichia coli clamp loader, is not required to open PCNA. The Rfc5 subunit is required to open PCNA. Consistent with this result, Rfc2·3·4·5 and Rfc2·5 subassemblies are capable of opening and unloading PCNA from circular DNA. Rfc5 is positioned opposite the PCNA interface from Rfc1, and therefore, its action with Rfc2 in opening PCNA indicates that PCNA is opened from the opposite side of the interface that the E. coli δ wrench acts upon. This marks a significant departure in the mechanism of eukaryotic and prokaryotic clamp loaders. Interestingly, the Rad·RFC DNA damage checkpoint clamp loader unloads PCNA clamps from DNA. We propose that Rad·RFC may clear PCNA from DNA to facilitate shutdown of replication in the face of DNA damage.

Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation

We have reconstituted a eukaryotic leading/lagging strand replisome comprising 31 distinct polypeptides. This study identifies a process unprecedented in bacterial replisomes. While bacteria and phage simply recruit polymerases to the fork, we find that suppression mechanisms are used to position the distinct eukaryotic polymerases on their respective strands. Hence, Pol ε is active with CMG on the leading strand, but it is unable to function on the lagging strand, even when Pol δ is not present. Conversely, Pol δ-PCNA is the only enzyme capable of extending Okazaki fragments in the presence of Pols ε and α. We have shown earlier that Pol δ-PCNA is suppressed on the leading strand with CMG (Georgescu et al., 2014). We propose that CMG, the 11-subunit helicase, is responsible for one or both of these suppression mechanisms that spatially control polymerase occupancy at the fork. DOI: http://dx.doi.org/10.7554/eLife.04988.001

Cost of rNTP/dNTP pool imbalance at the replication fork

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.

The replication factor C clamp loader requires arginine finger sensors to drive DNA binding and proliferating cell nuclear antigen loading.

Replication factor C (RFC) is an AAA+ heteropentamer that couples the energy of ATP binding and hydrolysis to the loading of the DNA polymerase processivity clamp, proliferating cell nuclear antigen (PCNA), onto DNA. RFC consists of five subunits in a spiral arrangement (RFC-A, -B, -C, -D, and -E, corresponding to subunits RFC1, RFC4, RFC3, RFC2, and RFC5, respectively). The RFC subunits are AAA+ family proteins and the complex contains four ATP sites (sites A, B, C, and D) located at subunit interfaces. In each ATP site, an arginine residue from one subunit is located near the γ-phosphate of ATP bound in the adjacent subunit. These arginines act as “arginine fingers” that can potentially perform two functions: sensing that ATP is bound and catalyzing ATP hydrolysis. In this study, the arginine fingers in RFC were mutated to examine the steps in the PCNA loading mechanism that occur after RFC binds ATP. This report finds that the ATP sites of RFC function in distinct steps during loading of PCNA onto DNA. ATP binding to RFC powers recruitment and opening of PCNA and activates a γ-phosphate sensor in ATP site C that promotes DNA association. ATP hydrolysis in site D is uniquely stimulated by PCNA, and we propose that this event is coupled to PCNA closure around DNA, which starts an ordered hydrolysis around the ring. PCNA closure severs contact to RFC subunits D and E (RFC2 and RFC5), and the γ-phosphate sensor of ATP site C is switched off, resulting in low affinity of RFC for DNA and ejection of RFC from the site of PCNA loading.

Mechanism of polymerase collision release from sliding clamps on the lagging strand

Replicative polymerases are tethered to DNA by sliding clamps for processive DNA synthesis. Despite attachment to a sliding clamp, the polymerase on the lagging strand must cycle on and off DNA for each Okazaki fragment. In the ‘collision release’ model, the lagging strand polymerase collides with the 5′ terminus of an earlier completed fragment, which triggers it to release from DNA and from the clamp. This report examines the mechanism of collision release by the Escherichia coli Pol III polymerase. We find that collision with a 5′ terminus does not trigger polymerase release. Instead, the loss of ssDNA on filling in a fragment triggers polymerase to release from the clamp and DNA. Two ssDNA‐binding elements are involved, the τ subunit of the clamp loader complex and an OB domain within the DNA polymerase itself. The τ subunit acts as a switch to enhance polymerase binding at a primed site but not at a nick. The OB domain acts as a sensor that regulates the affinity of Pol III to the clamp in the presence of ssDNA.

The RFC Clamp Loader: Structure and Function

The eukaryotic RFC clamp loader couples the energy of ATP hydrolysis to open and close the circular PCNA sliding clamp onto primed sites for use by DNA polymerases and repair factors. Structural studies reveal clamp loaders to be heteropentamers. Each subunit contains a region of homology to AAA+ proteins that defines two domains. The AAA+ domains form a right-handed spiral upon binding ATP. This spiral arrangement generates a DNA binding site within the center of RFC. DNA enters the central chamber through a gap between the AAA+ domains of two subunits. Specificity for a primed template junction is achieved by a third domain that blocks DNA, forcing it to bend sharply. Thus only DNA with a flexible joint can bind the central chamber. DNA entry also requires a slot in the PCNA clamp, which is opened upon binding the AAA+ domains of the clamp loader. ATP hydrolysis enables clamp closing and ejection of RFC, completing the clamp loading reaction.

SnapShot: The replisome.

In all organisms, successful cell division requires accurate copying of chromosomal DNA. To duplicate their genomes, all cells use a multiprotein apparatus known as the replisome (reviewed by Benkovic et al., 2001; McHenry, 2003; Yao and O’Donnell, 2009). The fundamental components of the replisome are conserved across viruses, bacteria, archaea, and eukaryotes (Table). They include a helicase to unwind the double-stranded DNA, a polymerase(s) to synthesize new strands of DNA, and a clamp loader to organize the complex on the DNA. The replisome assemblies at a region of the DNA, called the replication fork, where the double-stranded DNA is separated into two individual strands, which are both subsequently copied in the 5′ to 3′ direction of the DNA. In this SnapShot, we compare the specific components of the replisome in Escherichia coli with those of the replisome in eukaryotes. In addition, we describe how the lagging strand is synthesized from Okazaki fragments. Table