Mike O’Donnell

Trading Places on DNA—A Three-Point Switch Underlies Primer Handoff from Primase to the Replicative DNA Polymerase

This study reports a primase-to-polymerase switch in E. coli that closely links primase action with extension by DNA polymerase III holoenzyme. We find that primase tightly grips its RNA primer, protecting it from the action of other proteins. However, primase must be displaced before the beta sliding clamp can be assembled on the primed site. A single subunit of the holoenzyme, chi, is dedicated to this primase displacement task. The displacement mechanism depends on a third protein, SSB. Primase requires contact to SSB for its grip on the primed site. The chi subunit also binds SSB, upon which the primase-to-SSB contact is destabilized leading to dissociation of primase and assembly of beta onto the RNA primer. The conservation of this three-point switch, in which two proteins exchange places on DNA via mutually exclusive interaction with a third protein, is discussed.

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.

DNA polymerase clamp loaders and DNA recognition

Clamp loaders are heteropentameric ATPase assemblies that load sliding clamps onto DNA and are critical for processive DNA replication. The DNA targets for clamp loading are double‐stranded/single‐stranded junctions with recessed 3′ ends (primer‐template junctions). Here, we briefly review the crystal structures of clamp loader complexes and the insights they have provided into the mechanism of the clamp loading process.

Overproduction and analysis of eukaryotic multiprotein complexes in Escherichia coli using a dual-vector strategy.

Biochemical studies of eukaryotic proteins are often constrained by low availability of these typically large, multicomponent protein complexes in pure form. Escherichia coli is a commonly used host for large-scale protein production; however, its utility for eukaryotic protein production is limited because of problems associated with transcription, translation, and proper folding of proteins. Here we describe the development and testing of pLANT, a vector that addresses many of these problems simultaneously. The pLANT vector contains a T7 promoter-controlled expression unit, a p15A origin of replication, and genes for rare transfer RNAs and kanamycin resistance. Thus, the pLANT vector can be used in combination with the pET vector to coexpress multiple proteins in E. coli. Using this approach, we have successfully produced high-milligram quantities of two different Saccharomyces cerevisiae complexes in E. coli: the heterodimeric Msh2-Msh6 mismatch repair protein (248kDa) and the five-subunit replication factor C clamp loader (250 kDa). Quantitative analyses indicate that these proteins are fully active, affirming the utility of pLANT+pET-based production of eukaryotic proteins in E. coli for in vitro studies of their structure and function.

Replication Bypass of the Acrolein-Mediated Deoxyguanine DNA- Peptide Cross-links by DNA polymerases of the DinB Family

DNA-protein cross-links (adducts) are formed in cellular DNA under a variety of conditions, particularly following exposure to an alpha,beta-unsaturated aldehyde, acrolein. DNA-protein cross-links are subject to repair or damage-tolerance processes. These adducts serve as substrates for proteolytic degradation, yielding DNA-peptide lesions that have been shown to be actively repaired by the nucleotide excision repair complex. Alternatively, DNA-peptide cross-links can be subjected to replication bypass. We present new evidence about the capabilities of DNA polymerases to synthesize DNA past such cross-links. DNAs were constructed with site-specific cross-links, in which either a tetrapeptide or a dodecylpeptide was covalently attached at the N (2) position of guanine via an acrolein adduct, and replication bypass assays were carried out with members of the DinB family of polymerases, human polymerase (pol) kappa, Escherichia coli pol IV, and various E. coli polymerases that do not belong to the DinB family. Pol kappa was able to catalyze both the incorporation and the extension steps with an efficiency that was qualitatively indistinguishable from control (undamaged) substrates. Fidelity was comparable on all of these substrates, suggesting that pol kappa would have a role in the low mutation frequency associated with replication of these adducts in mammalian cells. When the E. coli orthologue of pol kappa, damage-inducible DNA polymerase, pol IV, was analyzed on the same substrates, pause sites were detected opposite and three nucleotides beyond the site of the lesion, with incorporation opposite the lesion being accurate. In contrast, neither E. coli replicative polymerase, pol III, nor E. coli damage-inducible polymerases, pol II and pol V, could efficiently incorporate a nucleotide opposite the DNA-peptide cross-links. Consistent with a role for pol IV in tolerance of these lesions, the replication efficiency of DNAs containing DNA-peptide cross-links was greatly reduced in pol IV-deficient cells. Collectively, these data indicate an important role for the DinB family of polymerases in tolerance mechanisms of N (2)-guanine-linked DNA-peptide cross-links.

Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation.

Significance All cellular life forms use a ring-shaped hexameric helicase during DNA replication. CMG (Cdc45, Mcm2–7, GINS) is the eukaryotic replicative helicase. CMG contains the ring-shaped hexameric Mcm2–7 that harbors the helicase motors. CMG is known to bind many other proteins, including a leading and lagging polymerase and primase. Thus, the threading of DNA through the CMG helicase at a replication fork determines the orientation of the associated polymerases at the replication fork, an important structural feature with many consequences that may direct future experimentation. This report uses cryo-EM single-particle reconstruction to image CMG that motored to a block site at a forked junction, enabling direct visualization of DNA threading through CMG. The eukaryotic CMG (Cdc45, Mcm2–7, GINS) helicase consists of the Mcm2–7 hexameric ring along with five accessory factors. The Mcm2–7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EM single-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork. The duplex stem penetrates into the central channel of the N-tier and the unwound leading single-strand DNA traverses the channel through the N-tier into the C-tier motor, 5′-3′ through CMG. Therefore, the N-tier ring is pushed ahead by the C-tier ring during CMG translocation, opposite the currently accepted polarity. The polarity of the N-tier ahead of the C-tier places the leading Pol ε below CMG and Pol α-primase at the top of CMG at the replication fork. Surprisingly, the new N-tier to C-tier polarity of translocation reveals an unforeseen quality-control mechanism at the origin. Thus, upon assembly of head-to-head CMGs that encircle double-stranded DNA at the origin, the two CMGs must pass one another to leave the origin and both must remodel onto opposite strands of single-stranded DNA to do so. We propose that head-to-head motors may generate energy that underlies initial melting at the origin.

A proposal: Evolution of PCNA's role as a marker of newly replicated DNA

Processivity clamps that hold DNA polymerases to DNA for processivity were the first proteins known to encircle the DNA duplex. At the time, polymerase processivity was thought to be the only function of ring shaped processivity clamps. But studies from many laboratories have identified numerous proteins that bind and function with sliding clamps. Among these processes are mismatch repair and nucleosome assembly. Interestingly, there exist polymerases that are highly processive and do not require clamps. Hence, DNA polymerase processivity does not intrinsically require that sliding clamps evolved for this purpose. We propose that polymerases evolved to require clamps as a way of ensuring that clamps are deposited on newly replicated DNA. These clamps are then used on the newly replicated daughter strands, for processes important to genomic integrity, such as mismatch repair and the assembly of nucleosomes to maintain epigenetic states of replicating cells during development.

A structural role for the PHP domain in E. coli DNA polymerase III

BackgroundIn addition to the core catalytic machinery, bacterial replicative DNA polymerases contain a Polymerase and Histidinol Phosphatase (PHP) domain whose function is not entirely understood. The PHP domains of some bacterial replicases are active metal-dependent nucleases that may play a role in proofreading. In E. coli DNA polymerase III, however, the PHP domain has lost several metal-coordinating residues and is likely to be catalytically inactive.ResultsGenomic searches show that the loss of metal-coordinating residues in polymerase PHP domains is likely to have coevolved with the presence of a separate proofreading exonuclease that works with the polymerase. Although the E. coli Pol III PHP domain has lost metal-coordinating residues, the structure of the domain has been conserved to a remarkable degree when compared to that of metal-binding PHP domains. This is demonstrated by our ability to restore metal binding with only three point mutations, as confirmed by the metal-bound crystal structure of this mutant determined at 2.9 Å resolution. We also show that Pol III, a large multi-domain protein, unfolds cooperatively and that mutations in the degenerate metal-binding site of the PHP domain decrease the overall stability of Pol III and reduce its activity.ConclusionsWhile the presence of a PHP domain in replicative bacterial polymerases is strictly conserved, its ability to coordinate metals and to perform proofreading exonuclease activity is not, suggesting additional non-enzymatic roles for the domain. Our results show that the PHP domain is a major structural element in Pol III and its integrity modulates both the stability and activity of the polymerase.

Replisome structure and conformational dynamics underlie fork progression past obstacles.

Replisomes are multiprotein complexes that unzip the parental helix and duplicate the separated strands during genome replication. The antiparallel structure of DNA poses unique geometric constraints to the process, and the replisome has evolved unique dynamic features that solve this problem. Interestingly, the solution to duplex DNA replication has been co-opted to solve many other important problems that replisomes must contend with during the duplication of long chromosomes. For example, along its path the replisome will encounter lesions and DNA-bound proteins. Recent studies show that the replisome can circumvent lesions on either strand, using the strategy normally applied to the lagging strand synthesis. Circumventing lesions can also be assisted by other proteins that transiently become a part of the replisome. The replisome must also contend with DNA-binding proteins and recent studies reveal a fascinating process that enables it to bypass RNA polymerase without stopping.

New insights into replisome fluidity during chromosome replication.

Several paradigm shifting advances have recently been made on the composition and function of the chromosomal DNA replication machinery. Replisomes appear to be more fluid and dynamic than ever imagined, enabling rapid and efficient bypass of roadblocks and template lesions while faithfully replicating chromosomal DNA. This fluidity is determined by many layers of regulation, which reach beyond the role of replisome components themselves. In fact, recent studies show that additional polymerases, post-transcriptional modifications, and chromatin structure are required for complete chromosome duplication. Many of these factors are involved with the more complex events that take place during lagging-strand synthesis. These, and other recent discoveries, are the focus of this review.