Significance All cellular life forms use a ring-shaped hexameric helicase during DNA replication. CMG (Cdc45, Mcm2–7, GINS) is the eukaryotic replicative helicase. contains Mcm2–7 that harbors motors. known to bind many other proteins, including leading and lagging polymerase primase. Thus, threading of through at replication fork determines orientation associated polymerases fork, an important structural feature with consequences may direct future experimentation. This report uses cryo-EM...

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 unwind double helix and synthesize two new DNA strands complementary parental strands. The details replisome function have been worked for bacteria but are much less well understood eukaryotic cells. We developed system studying in vitro using purified proteins. Using this system, we identified direct interaction...

We have reconstituted a eukaryotic leading/lagging strand replisome comprising 31 distinct polypeptides. This study identifies process unprecedented in bacterial replisomes. While bacteria and phage simply recruit polymerases to the fork, we find that suppression mechanisms are used position on their respective strands. Hence, Pol ε is active with CMG leading strand, but it unable function lagging even when δ not present. Conversely, δ-PCNA only enzyme capable of extending Okazaki fragments...

Replicative helicases in all cell types are hexameric rings that unwind DNA by steric exclusion which the helicase encircles tracking strand only and excludes other from ring. This mode of translocation allows to bypass blocks on is excluded central channel. Unlike replicative helicases, eukaryotic CMG partially duplex at a forked junction stopped block non-tracking (lagging) strand. report demonstrates Mcm10, an essential replication protein unique eukaryotes, binds greatly stimulates its...

Abstract The eukaryotic leading strand DNA polymerase (Pol) ε contains 4 subunits, Pol2, Dpb2, Dpb3 and Dpb4. Pol2 is a fusion of two B-family Pols; the N-terminal Pol module catalytic C-terminal non-catalytic. Despite extensive efforts, there no atomic structure for holoenzyme, critical to understanding how synthesis coordinated with unwinding path through CMG helicase-Pol ε-PCNA clamp. We show here 3.5-Å cryo-EM yeast revealing that Dpb3–Dpb4 subunits bridge modules holding them rigid....

Abstract High-resolution structures have not been reported for replicative helicases at a replication fork atomic resolution, prerequisite to understanding the unwinding mechanism. The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase contains Mcm2-7 motor ring, with N-tier ring in front and C-tier behind. is structurally divided into zinc finger (ZF) sub-ring followed by oligosaccharide/oligonucleotide-binding (OB) fold ring. Here we report cryo-EM structure of on forked DNA 3.9 Å, revealing...

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

DNA polymerases attach to the sliding clamp through a common overlapping binding site. We identify small-molecule compound that binds protein-binding site in Escherichia coli beta-clamp and differentially affects activity of II, III, IV. To understand molecular basis this discrimination, cocrystal structure chemical inhibitor is solved complex with beta compared structures Pol IV peptides bound beta. The analysis reveals small molecule localizes region which different ways. results suggest...

The current view is that eukaryotic replisomes are independent. Here we show Ctf4 tightly dimerizes CMG helicase, with an extensive interface involving Psf2, Cdc45, and Sld5. Interestingly, binds only one Pol α-primase. Thus, may have evolved as a trimer to organize two helicases α-primase into replication factory. In the 2CMG–Ctf43–1Pol factory model, CMGs nearly face each other, placing lagging strands toward center leading out sides. single centrally located prime both sister replisomes....

Significance The structure of the eukaryotic chromosomal replicase, DNA polymerase (Pol) δ, was determined in complex with its cognate proliferating cell nuclear antigen (PCNA) sliding clamp on primed DNA. results show that Pol3 catalytic subunit binds atop PCNA ring, and two regulatory subunits Pol Pol31, Pol32, are positioned off to side clamp. such as thread straight through circular Considering large diameter clamp, there is room for water between inner walls PCNA, indicating...

The proliferating cell nuclear antigen (PCNA) clamp encircles DNA to hold polymerases (Pols) for processivity. Ctf18-RFC PCNA loader, a replication factor C (RFC) variant, is specific the leading-strand Pol (Polε). We reveal here underlying mechanism of specificity Polε using cryo-electron microscopy and biochemical studies. found that both contain structural features direct loading onto DNA. Unlike other loaders, has disordered ATPase associated with diverse cellular activities (AAA+) motor...

The 9-1-1 DNA checkpoint clamp is loaded onto 5'-recessed to activate the damage that arrests cell cycle. a heterotrimeric ring in Saccharomyces cerevisiae by Rad24-RFC (hRAD17-RFC), an alternate loader which Rad24 replaces Rfc1 RFC1-5 of proliferating nuclear antigen (PCNA). loading mechanism has been mystery, because, unlike RFC, loads PCNA 3'-recessed junction, junction. Here we report two cryo-EM structures Rad24-RFC-DNA with closed or 27-Å open clamp. reveal completely unexpected can be...

Abstract The eukaryotic polymerase α (Pol α) synthesizes an RNA-DNA hybrid primer of 20–30 nucleotides. Pol is composed Pol1, Pol12, Primase 1 (Pri1), and Pri2. Pol1 Pri1 contain the DNA RNA primase activities, respectively. It has been unclear how hands over from to for extension, length defined. Here we report cryo-EM analysis yeast in apo, initiation, elongation, hand-off extension states, revealing a series very large movements. We reveal critical point at which Pol1-core moves take...

Chromosomal DNA polymerases are tethered to by a circular sliding clamp for high processivity. However, lagging strand synthesis requires the polymerase rapidly dissociate on finishing each Okazaki fragment. The Escherichia coli replicase contains subunit (tau) that promotes separation of from its segments. This report reveals mechanism this process. We find tau binds C-terminal residues polymerase. Surprisingly, same "tail" interacts with beta clamp, and competes sequence. Moreover, acts as...