The Replication Fork and Replisome

The Replication Fork and Replisome
Lecture 1: DNA Polymerase Use of biochemistry (assays) and genetics (phenotypes) to define function Fidelity/Specificity: bioregulation through substrate control of molecular choice Lecture 2: The Replication Fork and Replisome Breaking down complex processes into intermediates and subreactions In vivo and in vitro analysis of players, intermediates, and activities Defining activity dependencies to understand their order and timing General principles from first and second lecture

Dissecting Complex Molecular Mechanisms
Slide shows examples of the molecular mechanisms and pathways that you are used to seeing in textbooks Today’s lecture will discuss the tools and strategies used to come up with these mechanisms. This will allow you to evalutate what aspects of these diagrams are actually supported by experiments and what parts are just artistic license or unwarranted scientific assumptions.

I4………….. In A1 A2 A3 A4 An+1 A1 A2 S = substrate S I P P = product I = intermediate A = activity DNA Polymerase III 5’ 3’ In most molecular biological processes a substrate is converted to a product through a series of intermediates. To understand the process we need to identify the intermediates. The transition from one intermediate to the next then define the steps and activities needed for the process to occur. As more intermediates are identified, more refined and specific steps and activities are defined. To illustrate this scientific approach we will show how the replication of a parental DNA molecule to form daughter molecules can be broken down into a series of steps From the last lecture we know that somehow the parental DNA is converted to daughter DNA copies via a process that requires DNA polymerase But obviously there is much more involved than one enzyme, In this lecture, we will see how a replication bubble with two replication forks was shown to be a fundamental intermediate in the replication process Then, we will see how more specific intermediates, steps, and activities functioning at the replication fork were identified.

I4………….. In A1 A2 A3 A4 An+1 S = substrate S P P = product I = intermediate A = activity A new intermediate must be: methods to distinguish molecular structures 1) structurally (or functionally) distinct 2) derived from the starting substrate Since the key to defining molecular mechanisms is identifying the intermediates in a molecular pathway, let me first enumerate the criteria for identifying a new intermediate The intermediate must be: 1) structurally distinct from substrate, product, and other known intermediates. (for example, a new assembly of proteins or a different nucleic acid conformation) 2) derived from the starting substrate. 3) converted to the product These criteria in turn highlight two types of experimental methods that are necessary to identify new intermediates methods to distinguish molecular structures methods to follow molecular fate Moreover, these methods must often deal with intermediates that are transient or in low abundance methods to follow molecular fate 3) converted to the final product

I4………….. In A1 A2 A3 A4 An+1 S = substrate S P P = product I = intermediate A = activity How to structurally characterize intermediates? How to detect intermediates? How to identify the proteins/nucleic acids responsible for the activities? Detecting highly abundant intermediates by precursor labeling Direct visualization of single molecules by microscopy I discuss these experimental methods by addressing the following questions How does one detect intermediates and show they are on a molecular pathway from subtrate to product How does one structurally characterize intermediates How does one identify the protein and/or nucleic acid machinery that provide the activity, so that we can understand how this machinery works. These questions will hopefully provide a conceptual framework that you can use for understanding a mechanistic paper or designing experiments for your proposal. Before I go into a more systematic discussion of these approaches, I will illustrate the first two using the early efforts by John Cairns to understand what a DNA replication intermediate might look like in E. coli It should be noted that in rapidly growing bacteria, most DNA molecules are actively replicating, so that these replication intermediates are in high abundance To detect these replication intermediates Cairns labeled them using radioactive nuclotide precursors And to structurally characterize them he looked at single molecules under the microscope

Visualization of E. coli DNA Replication Intermediates
Label E. coli ~ 2 generations with radioactive thymidine (T) daughter parent E. coli genome is circular and replicates with a replication bubble containing two daughter arms connected at each end to the remaining unreplicated parental segment Gently lyse cells and let DNA settle and stick onto a membrane fork Tt TT DNA replication is localized to two moving replication forks that travel bidirectionally around the molecule probably from a defined site of initiation Autoradiograph with coating of photographic emulsion Develop emulsion and analyze DNA structures under microscope, The experiment is outlined on the left, but because of lack of time, I leave you to read the experimental details and the precise interpretation of the results in the comments on my powerpoint presentation. Suffice it to say the structural analysis of replication intermediates like the one pictured allowed Cairns to conclude that: 1) The circular E. coli genome replicates as a replication bubble with two newly synthesized daughter arms connected at each end with the parental DNA 2) DNA replication is localized to two moving replication forks that proceed bidirectionally away from a site of initiation Although the replication intermediates Cairns wanted to see were very abundant, they were also extremely fragile, so the challenge was developing a way to isolate them intact. He incorporated tritiated thymidine (sugar plus base with no phosphates) in E. coli for about 2 generations, gently lysed cells, let DNA molecules settle and stick onto a membrane, covered the membrane with photographic emulsion, then developed the autoradiograph and examined it under a microscope. The incorporated radioactive thymidine allowed him to detect the DNA strands of replicating molecules as silver grains on the emulsion He could monitor DNA shape, measure DNA lengths, and, by quantifying grain densities, determine if one or both strands were labeled. What is shown is a large (4.5 million bp) circular genome with a replication bubble comprised of two equal length daughter arms accounting for 2/3 of the genome and one single unreplicated parental arm containing the remaining 1/3. Other molecules showed replication bubbles of different sizes, suggesting that the bubbles expand outward at these forks due to new DNA synthesis at the forks. Thus, these results suggest that DNA replication is localized to moving replication forks. It should be noted that the question of molecular fate was addressed by the radioactive strand labeling analysis, analagous to how density strand labeling was used to address molecular fate in the Meselson Stahl experiment. As explained in more detail in the Appendix, one could infer that a mostly HL parental genome was being replicated to generate one HH daughter and one HL daughter in this intermediate. ----- Meeting Notes (1/9/17 10:06) ----- Analyze: - Shapes - Lengths - Grain Density double-strand labeling (TT) vs single-strand labeling (Tt)

A key replication intermediate: the replication bubble
How to detect intermediates? Detecting highly abundant intermediates by precursor labeling Direct visualization of single molecules by microscopy How to structurally characterize intermediates? IN summary, Cairn’s use of thymidine labeling and microscopy to structurally characterize replicating E. coli genomes identified a key replication intermediate: the replication bubble with bidirectional replication forks How to identify the proteins/nucleic acids responsible for the activities?

How is DNA synthesized at the replication fork?
requires 3’>5’ polymerase 5’ 3’ requires periodic nicking at fork 5’ 3’ These studies raised the question of exactly how is DNA synthesized at the replication fork? One could imagine several models, each with different requirements and structural predictions for the replication intermediates at the fork Fully continuous Strand switching Semidiscontinuous 4) Fully discontinuous What we now know is the correct model, the semidiscontinuous model, has two predictions about replication fork intermediates. there will be transient short daughter DNA intermediate that become incorporated into larger daughter molecules there will be short SS parental segments on one arm requires periodic priming and ligation 5’ 3’ requires periodic priming and ligation 5’ 3’ transient short daughter DNA intermediate short SS parental segments on one arm

? How to detect intermediates? How to structurally characterize intermediates? Before we examine how these fork intermediates were examined I want to provide an overview of three classic strategies to detect intermediates in a molecular reaction, especially when they are transient and/or rare You will see these strategies used over and over again in different guises in papers, seminars and hopefully in your proposals. How to identify the proteins/nucleic acids responsible for the activities?

To Transiently Enrich Successive Intermediates
Detecting Intermediates Synchronize Reaction To Transiently Enrich Successive Intermediates Block Reaction Step To Accumulate Intermediate S P I1 I2 Examples of blocks: - remove/inactivate protein - remove cofactor - lower temperature - add inhibitor { Partial Reaction Pulse-Chase Label a Synchronous Cohort Label can sensitivity of detection Molecular fate established by chase time Molecular fate suggested by block and possibly established if block can be reversed time S S I1 I2 I1 I2 P S The first strategy is to synchronize the reaction and observe the possible intermediates that appears at different times. For example, in a the test tube synchronization occurs automatically when you finally add together all the components needed for a reaction At the beginning the starting substrate is the primary species, but as the reaction proceeds the substrate will disappear and early intermediates predominate, followed by later intermediates, and eventually the product. This method can enrich for transient and scarce intermediates. The idea is to sample the reaction at different times and characterize the nature of the intermediates as a function of time. The successive waves of different molecules, I.e. the disappearance of one species coinciding with the appearance of another, provides a temporal order of molecules and strongly suggests a path of molecular fates. However, it falls short of establishing molecular fate because it doesn’t truly demonstrate that one population of molecules was indeed transformed into the next. There are also several potential limitations to this synchronization approach 1) The synchronization procedure may perturb the reaction in a way that might mislead you. 2) The tightness of the synchrony limits the temporal resolution of the experiment, i.e. your ability to order intermediates and steps that occur extremely fast; for example, if you synchronous population consists of molecules that are within a minute of each other on the pathway, you will not be able to order intermediates that transition from one to another in less than a minute. 3) Synchrony is always degraded with increasing time. Note that real time observation of single molecules (or single cels) essentially follow this strategy but have two powerful advantages: -- there is no need to synchronize a population of molecules -- because you are observing the same molecule continuously, molecular fate can be established and not just inferred There is still limitation on the temporal resolution of the experiment but now that limitation is no longer governed by how tightly the population can be synchronized. It is determined by how frequently you can monitor the structural feature distinguishing intermediate states. Since single molecules are often monitored microscopically, this frequency is often determined by how many pictures per unit time can be recorded by your video camera. P I2 I1 Molecular fate implied by sequential appearance and disappearance of molecules Real time observation of single molecules use this strategy but do not require synchronization - can establish molecular fate

To Transiently Enrich Successive Intermediates
Detecting Intermediates Synchronize Reaction To Transiently Enrich Successive Intermediates Pulse-Chase Label a Synchronous Cohort Block Reaction Step To Accumulate Intermediate S P I1 I2 Examples of blocks: - remove/inactivate protein - remove cofactor - lower temperature - add inhibitor { Partial Reaction Molecular fate suggested by block and possibly established if block can be reversed time S P I2 I1 time S S P I1 I2 S I1 I2 I1 I2 P S P I1 I2 P I2 S I1 I2 P A second classic strategy for studying intermediates is to do a Pulse-Chase experiment. Labeled precursors are incorporated into a population of intermediate molecules for a short pulse of time, then further incorporation is prevented by greatly diluting out the label with unlabeled precursor during a chase period. By using a short labeling pulse, one effectively labels a synchronous cohort of molecules in an unsynchronized population and can monitor the fate of this cohort. With increasing chase time different intermediate populations and eventually the product gets labeled. Following the label during the chase allows one to connect the molecular fates of different intermediates and demonstrate that they are indeed intermediates because they get transformed into the final product. The use of the label (for example radioactivity or a fluorescent probe) can also enhance the sensitivity and specificity of detection for scarce intermediates The longer one labels during th pulse the more sensitive your assay, but you tradeoff is less synchrony of your labeled cohort. So usually the pulse is done for the shortest time that will give you good signal to noise sensitivity., The Meselson and Stahl paper that you will read for discussion uses a variation of this strategy to follow the fate of both parental strands and nucleotide precursors during DNA replication. Molecular fate implied by sequential appearance and disappearance of molecules Molecular fate established by chase Real time observation of single molecules use this strategy but do not require synchronization - can establish molecular fate Label can enhance sensitivity and specificity of detection

{ Detecting Intermediates S I1 I2 P I1 S P P S P S S P S P S P
Synchronize Reaction To Transiently Enrich Successive Intermediates Pulse-Chase Label a Synchronous Cohort Block Reaction Step To Accumulate Intermediate time S P I2 I1 time S P S P I1 I2 S I1 I2 S I1 I2 S I1 P { I1 I2 P S P I1 I2 Partial Reaction P I2 S I1 I2 P The third approach for studying intermediates is to temporarily block a molecular pathway For example, if the reaction occurs in a test tube with defined components, one can leave out one of the components The block dams up the pathways and allows putative intermediates to accumulate to levels that are easier to detect and study The block also defines a partial reaction consisting of all the steps before the block The molecular fate of the accumulated molecules is suggested by the absence of the product However one can directly establish these molecules become the product, if one can reverse the block and see the former chased into the latter. Molecular fate implied by sequential appearance and disappearance of molecules Molecular fate established by chase Molecular fate suggested by block and established if reversing block converts I to P Label can enhance sensitivity and specificity of detection Real time observation of single molecules use this strategy but do not require synchronization - can establish molecular fate

How to detect intermediates? Detect by pulse labeling Size analysis on alkaline sucrose gradients How to structurally characterize intermediates? We are now going to see how a variant of the pulse-chase strategy followed by size analysis of the labeled intermediate led to the first indication that there might be a transient short DNA synthesis intermediate at the fork. 5’ 3’ transient short daughter DNA intermediate

Detection and Analysis of Newly Synthesized DNA
Pulse label asynchronously replicating E. coli with H-thymidine 3 Extract DNA and alkali denature In this famous experiment, Okazaki pulse labeled asynchronously replicating E. coli for a variety of short times, separated labeled daughter strands from unlabeled parental strands by alkali denaturation, and size fractionated the strands by alkanline sucrose gradient. Plotted here is the amount of radioactive label versus increasing fragment size In an asynchronous populations, most of the nascent daughter strands on both leading and lagging arms are already quite large. Hence, if we were just extending off of these nascent strand, we would expect all the most recent incorporation to be associated with these large nascent strands. However, the shortest pulse labeled DNA, which represents the most recent incorporation of nucleotides, is mostly in small molecules. Longer pulse labeled DNA is much larger, raising the possibilty that the short molecules become incorporated into larger molecules, presumably through ligation. Later detailed structural analysis of these okazaki fragments by other labs demonstrated that there was an 8-10 nt RNA segment at the 5’ end linked by 5’ to 3’ phosphodiester bond to the DNA. In the problem set next week, some of you will be asked to analyze Okazaki’s experiments in more detail, because the conclusions you can draw from his results are not as simple and straightforward as I have presented here. Centrifuge in alkaline sucrose gradient to separate by size Measure radioactivity in gradient fractions (increasing size ) The newest DNA synthesized is mostly small (~ bp) Structural analysis by others showed the newest DNA has 8-10 nt RNA linked 5’ > 3’ to 5’ end of DNA.

How to detect intermediates? Study abundant phage lambda replication intermediates Visualize DS vs SS structure at fork with EM How to structurally characterize intermediates? Another prediction from the semidiscontinuous model was that short SS parental segments would be present only on one daughter arm. This prediction was investigated by Schnos using highly abundant phage lambda replication intermediates and applying a high resolution EM analysis of forks that could distinguish double strand from single strand DNA 5’ 3’ short SS parental segments on one arm

Higher resolution visualization of replication fork structure
Inman & Schnos (1971): electron microscopy of replicating phage l DNA SS is often seen on only one arm of each fork In some cases interrupted by short DS segment DS SS DS SS DS SS 5’ 3’ 3’ 5’ What they saw was single-stranded regions near the fork on one daughter arm and almost never on both The size of the SS region, a couple of thousand nucleotides, was consistent with size of the discontinuous gaps expected to be filled in with okazaki fragments Often the SS region was interrupted with a DS region consistent with presence of a growing ozakaki fragment Interestingly, when both forks of a bubble had SS DNA, the SS DNA was always in trans (opposite daughter arms), as expected for the semidiscontinuous model because of the flipped polarity of the opposing forks

Semidiscontinuous DNA Synthesis
5’ 3’ Together these and other results indicated that DNA synthesis at replication forks occurs in a semidiscontinuous manner.

I4………….. In A1 A2 A3 A4 An+1 S = substrate S P P = product I = intermediate A = activity How to structurally characterize intermediates? How to detect and identify intermediates? How to identify the proteins/nucleic acids responsible for the activities? Before I discuss the ramifications of this model and how proteins and biochemical activities were introduced into the picture, let me give you a reference list of some of the ways that intermediates are structurally characterized

Interacting Sequences
Structural Analysis of Intermediates Examples of structural features that can be monitored Nucleic Acids Proteins Modifications Ligand Binding Conformation Covalent Linkages Cofactor (NTP) Status Complexes Composition Stoichiometry Conformation Interacting Sequences Interacting Domains Each of these features requires some assay to detect and quantify This list is not for you to memorize but for you to refer to when you read about experiments and when you design your own experiments for your proposal One can already recognize some of the features that have been used by Cairns, Okazaki, and Schnos in their experiments The experiments we discussed so far focused on the nucleic acid structure of replicating molecules, so I start with those. Cairns observed the size and shape of the replicating E. coli genome as well as the extent to which DS or SS DNA was labeled. Okazaki analyzed the size of the newly synthesized DNA, their DS versus SS character, and tried to assess their pairing status by comparing alkaline denaturing conditions to neutral conditions The analysis of RNA primers in Okazaki fragments depended on the characterization of primer size, covalent linkages, strand polarity, and the ability to distinguish deoxyribonucleotides from ribonucleotides. Finally, Schnos analyzed the size, shape, and DS versus SS character of replicating lambda forks, and could infer that the SS regions were always on strands of the same polarity. For most of the processes highlighted in this course, most of the intermediates a protein component as well as a nucleic acid component 2) For protein components one can often detect various types of alterations Some are quite straightforward, such as labeling with radioactive phosphate to determine whether the protein is phosphorylated. Others are much more challenging, such as detecting conformational changes by looking at altered accessibility of the protein to proteases 3) Finally, the structure of the intermediate as a complex between various protein and nucleic acid components can be assayed. Many processes involve the stepwise assembly of a large complex and various intermediates can be characterized just by changes in the composition, stoichiometry or conformation of proteins and nucleic acids in successive intermediates which can be monitored with chry of the most important types of changes, conformation, however, is often the hardest to monitor Finally, the full nature of the complex can changes various features. So if one intermediate has fully double stranded DNA bound by a protein, and a subsequent intermediate has a localized single stranded character with the protein bound by ATP, one might speculate that the ATP binding to the protein turned on an activity to unwind the DNA. Size Shape DS versus SS Strand Pairing Strand Polarity Covalent Linkages Modifications Topology Sequence

Semi-Discontinuous DNA Synthesis
Leading strand: polymerase moves continuously in same direction as replication fork Lagging strand: polymerase moves discontinuously in opposite direction as replication fork 5’ D 3’ C Lagging Fork Movement B 3’ A 5’ Leading 5’ 3’ In the semidiscontinuous model for DNA replication one can define a - leading strand, where the polymerase moves continuously in the same direction as the fork and - lagging strand, where the polymerase moves discontinuously in the opposite direction as the replication fork. The identification of sequential replication fork intermediates suggests a number of biochemical activities in addition to DNA polymerase that are needed to carry out DNA replication -- DNA unwinding and single-strand exposure -- RNA priming -- Replacement of RNA primer from nascent strand with DNA -- Ligation of okazaki fragment to nascent strand Additional activities inferred from replication intermediate analysis A. helix unwinding B. priming Okazaki fragment synthesis & processing prokaryotes: 1–2 kb eukaryotes: 100–200 bp C. primer replacement D. ligation

Understanding Molecular Mechanisms
P I1 I2 I3 I4………….. In A1 A2 A3 A4 An+1 S = substrate S P P = product I = intermediate A = activity How to detect intermediates? How to structurally characterize intermediates? How does one identify the proteins and/or nucleic acids that are responsible for the individual activities in a molecular mechanism? In the first lecture we discussed one approach. -- guess at an activity that might be needed, like nucleotide polymerization -- use trial and error to devise an assay for this activity -- look for and purify the activity -- generate a mutant in the gene for the protein to show it is important for the process being studied But this only gets a piece of the puzzle, and there may be many activities that we can’t guess at, or don’t know how to devise assays for. How do I study the complete cellular process in a manner that allows me to break it down into many individual activities that I can eventually purify and study biochemically? One very powerful approach is to have a soluble in vitro system that can recapitulate the process in a test tube. How to identify the proteins/nucleic acids responsible for the activities?

Using in vitro (soluble cell-free) Systems
The advantages of an in vitro system for understanding mechanism Purifying biochemical activities from in vitro systems Dissecting the purified system to understand its activities So I will now discuss: 1) The advantages of having an in vitro system for understanding cellular process 2) How one validates the in vitro system to provide confidence you are not studying an artifact 3) How one can purify activities required for the process without even knowing exactly what those activities are 4) How one can use the purified system to uncover the nature of specific activities

Advantages of an in vitro system to study mechanism
Can isolate a process from other competing or disruptive processes S P I1 I2 I3 I4………….. In A1 A2 A3 A4 An+1 S = substrate S P P = product I = intermediate A = activity How to detect and identify intermediates? Easier to synchronize, pulse-label, or block the process How to structurally characterize intermediates? First it is easier to study the process independent of competing, associated, or confounding processes. For example, concurrent repair processes can generate confounding repair intermediates that complicate the analysis of replication intermediates And regulatory mechanisms that might be restraining the process can be stripped away Second it easier to identify and detect intermediates in vitro It is easier to synchronize, pulse label, or block the process and it is easier to introduce defined intermediates to determine their molecular fate For example, if you are lucky enough to have the right mutant you can conditionally inactivate a protein in cells, but it is much easier to simply immunodeplete a protein from an in vitro system or leave it out of a purified system Third it is easier to structurally characterize intermediates in vitro Chemical, enzymatic, or immunologic probes are easier to introduce And the intermediates are usually in greater abundance and easier to isolate Fourth, one can separate and purify activities required for individual steps in a process. Importantly, with an invitro assay that recapitulates a complex biological process, one can purifiy individual activities needed for that process without knowing any details of that assay. Together all these advantages provide a powerful incentive to develop in vitro systems Note, that even without the use of the in vitro system to purify activities and study individual activities, there are many advantages to using such a system. Thus, some systems which have proven difficult to purify activities from, like the xenopus egg extract system to study DNA replication and the in vitro splicing systems, have still provided powerful extract systems for identifying intermediates and breaking down processes into individual steps. The problem is that developing in vitro systems can be extremely challenging, requiring lots of trial and error and often a good dose of luck. As we saw in lecture 1, the development of a DNA polymerase assay was extremely difficult and in vitro systems are essentially assays for complex molecular processes involving many activities. Nonetheless, as I will illustrate with DNA replication, there are enormous advantages to using an in vitro system for understanding molecular mechanisms. Easier to isolate and structurally analyze intermediates Easier to introduce various defined intermediates (or substrates) How to identify the proteins/nucleic acids responsible for the activities? Can separate and purify activities without any a priori knowledge about them

Fractionation & Reconstitution In Vitro Complementation
Purifying (undefined) biochemical activities from in vitro systems Fractionation & Reconstitution In Vitro Complementation Here I discuss how one can use an in vitro system to fractionate and purify all the activities required for a molecular process. Once you can reconstitute the entire process in vitro with purified activities, you have an extremely powerful system for identifying intermediates and the activities that convert one intermediate to another. The power of the approaches described here is that you can purify activities without knowing any details about them The assay for each activity is its ability to help reconstitute the full in vitro reaction when combined with the other required activities. There are two major ways to do this Fractionation and reconstitution As individual activities for a complex in vitro reaction are separated by various fractionation techniques, no individual fraction will display the full reaction activity But mixing the right fractions in the right proportion can reconstitute the full reaction Once this first separation and reconstitution is achieved, each individual fraction with reconstituting activity can be further fractionated based on this reconstitution assay. 2) In vitro complementation This approach requires the ability to specifically inactivate one activity in the extract Often this inactivation takes advantage of mutants obtained from a previous genetic analysis of the process, but it could also be a chemical or pharmacological inactivation. In this strategy one tries to rescue the inactivated extract by supplementing it with a fraction from the wild type extract. This rescuing activity then becomes the basis for purifying the activity that was inactivated. In both cases if the activity you are purifying starts to remind you of some activity that has already been purified, or if you can guess at the identity of the predominant protein coming through the purification, you can sometimes shortcut the purification process by seeing if the fraction can be replaced by the previously purified activity or recombinant proteins. Note: These fractionation purification schemes depend on systems that have readily separable activities that can reconstitute the full system when they are simply mixed together in a test tube The DNA replication machinery satisfies this requirement, but not all systems do. For example, the proteins and RNA that make up the ribosome are not readily separable, have little activity alone, and cannot be readily reconstituted into an active ribosome simply by mixing components in a test tube Slightly more separable is the splicing machinery, which can at least be separated and reconstituted at the level of large ribonucleoprotein snRNP complexes, but these components display little splicing activity until assembled into the full spliceosome, and so far is has not been possible to reconstitute snRNP complexes from purified components Hence, it has been more challenging to dissect the mechanisms of these ribonucleoprotein machines and there has been more dependence on the use of molecular genetic strategies to perturb the structure of these machines or substrates.

Phage T4 DNA Replication in vitro
Biochemical activities mostly purified by in vitro complementation Can reconstitute reaction with six “purified” activities One of the early in vitro replication systems was developed at UCSF in Bruce Albert’s lab. Phage T4 is an E. coli virus that replicates vigorously once it infects cells. A lot of genetic analysis of Phage T4 had been performed, so genes required for its DNA replication had already been defined by conditional mutants Extracts made from T4 infected E. coli were shown to be able to convert a SS circular DNA to a nicked circle and then to a rolling circle containing a fork with both a leading and lagging strand. The reaction was shown to have many of the characteristics and dependencies of T4 DNA replication in vivo. It wasn’t perfect, for example, okazaki fragments on the lagging strand were never processed to form a long continuous nascent strand. But it still provided a powerful system for understanding the assembly and action of a replication fork. Bruce Alberts was able to take advantage of the extensive genetics performed on phage T4 to purify most of the biochemical activities in his invitro system by in vitro complementation. This allowed him to replace the crude extract with a collection of six purified proteins each with an undefined activity. He labeled the proteins by the mutant used to purify their activity; do NOT memorize these numbers; it is only the activities that are important Later one of these “purified” protein, the gene 32 protein, was found to be contaminated with a seventh activity, that was subsequently purified away from the gene 32 protein.

41 is required for rapid strand displacement synthesis on DS DNA
A Helix Unwinding Activity (Helicase) 41 is required for rapid strand displacement synthesis on DS DNA The starting point in this set of experiments is a 6 protein reaction in which a protein, which Bruce had previously shown was required for priming DNA synthesis, has been left out. The remaining 6 proteins can perform rapid strand displacement synthesis starting with a nicked DS circle. The 3’ end of the nick can prime the continuous leading strand synthesis of the rolling circle fork, but the lagging strand synthesis is missing because okazaki fragments cannot be primed. When the gene 41 protein is removed, the reaction with the remaining five proteins is qualitatively the same, but quantitatively DNA synthesis is much slower and the displaced strands are much shorter. Thus the gene 41 protein is required for rapid strand displacement synthesis on DS DNA One possibility is that the gene 41 protein is needed to increase the activity and speed of the DNA polymerase. However, this is unlikely, because in the absence of the gene 41 protein, the remaining 5 proteins can rapidly synthesize DNA on single stranded templates. So simplest inference is that the gene 41 protein is only needed to help the replication machinery push through DS DNA Having a purified protein, also allows one to assay it for other standard activities. The gene 41 protein was found to have GTPase or ATPase activity and this activity was greatly stimulated by SS DNA To show that this hydrolytic activity is relevant to the first activity they disrupted the hydrolysis using a poorly hydrolyzed GTP analog and showed that strand displacement synthesis was greatly slowed. This led to the hypothesis that gene 41 protein uses the energy from ATP/GTP hydrolysis to help separate DNA strands at the replication fork And this in turn led to the development of a direct assay for strand separation coupled to nucleotide hydrolysis, i.e. a helicase activity? This assay starts with a labeled oligo that migrates very slowly in a gel because it is initially annealed to a larger piece of single stranded DNA. Displacement of the oligo by a helicase activity is detected by the much faster migration of the labeled oligo. Gene 41 protein was shown to have helicase activity SLOW no 41 A direct assay for helicase activity * FAST 41 is NOT required for rapid synthesis on SS DNA FAST no 41 41 has GTP/ATPase activity Greatly stimulated by SS DNA Inhibition by GTPS slows strand displacement synthesis

Replicative Helicases
Form hexameric rings that encircle single-stranded DNA and hydrolyze ATP to translocate unidirectionally along the DNA Turns out replicative helicases are very important in DNA replication and their loading onto the DNA is one of the earliest and most critical part of the initiation reaction Many replicative helicases form hexameric rings that can wrap around SS DNA and use nucleotide hydrolysis to translocate unidirectionally down the strand and peel away any hybridized DNA it runs into Prokaryotic and eukaryotic replicative helicases differ in the direction that they travel along SS DNA and thus which strand they travel along at the replication fork The diagram shows DnaB traveling along the lagging strand to push apart the parental strands The mechanisms of how helicases convert chemical energy to unidirectional translocation is now a subject of intense interest with people applying atomic structure and single molecule analysis to the problem These proteins belong to the family of AAA ATPases, which form oligomers with ATP binding pockets present in the interface of subunits. Such intersubunit bindings sites allows nucleotide binding and hydrolysis to be coupled to large conformational changes of the complex. Finally, I should mention that proteins whose primary structure resemble helicases participate in every process involving nucleic acids. Few have actually been shown to have helicase activity in vitro It is possible that its is because these helicases need just the right context to unlock their activity, or because maybe they are acting as translocases to displace proteins off DNA. This is a reminder that function is not defined by sequence homology but by specific assays or mutant phenotypes. Prokaryotes 5’ > 3’ (on lagging strand): DnaB Eukaryotes 3’ > 5’ (on leading strand): Cdc45-Mcm2-7-GINS 5’ 3’ DnaB Belong to AAA+ ATPases family, which form multimeric complexes and couple ATP binding and/or hydrolysis to conformational changes

Activities for okazaki fragment maturation
(E. coli) DNA Pol I (5’>3’ exo) Excise Primer DNA Pol I Fill-In Gap Ligase Seal Nick Fragments need to be stiched to gether but before that the primer needs to be replaced. WHY? Fidelity Primase can’t proofread because no primer requirement. Thus remove and replace Easier to distinguish by making it RNA instead of DNA

Replication Fork Tasks and Activities
Leading Strand Task Activity synthesize DNA polymerase separate parental strands helicase prime polymerase primase replace primer nuclease/polymerase connect okazaki fragments ligase stabilize SS DNA SSBP Through studies like these activities for various tasks that need to be performed at the replication fork have been identified. We have already discussed the top five tasks and activities Additional activities include: An activity to stabilize single-stranded DNA structure: single-stranded binding protein This activity protects SS DNA both from nuclease degradation and from base pairing to itself to form secondary structures that interfere with priming and polymerization. An activity to keep the DNA polymerase from falling off the template prematurely, which I will discuss next: clamp and clamp loader An activity to allow the separation of topologically linked parental DNA strands: topoisomerase, which I won’t have time to discuss ensure processivity clamp loader/clamp Lagging Strand unlink parental strands topoisomerase

Some activities may affect the rate, fidelity, specificity, or regulation of these steps S P I1 I2 I3 I4………….. In A1 A2 A3 A4 An+1 S = substrate S P P = product I = intermediate A = activity How to detect and identify intermediates? How to structurally characterize intermediates? Not all activities are absolutely required for a specific step in a biological process Many affect the rate, fidelity, specificty, or regulation of these steps One of the properties of DNA polymerases that affect their overall rate is their processivity How to identify the proteins/nucleic acids responsible for the activities?

Processivity How many times an enzyme can act repeatedly on a substrate before dissociating from it Assay: measure product size under conditions where an enzyme cannot reassociate with its substrate once it dissociates Condition 1: preload enzymes onto substrates then dilute Condition 2: excess substrate (e.g. primer-template) The processivity of an enzyme is a measure of the number of times an enzyme can act repeatedly on a substrate before dissociating It is inversely related to its rate of dissociation from its substrate Replicative DNA polymerases need to have very low off rates if they are to replicate large tracts of DNA There are two ways to assay processivity distributive polymerase (not processive) processive polymerase

An activity that enhances polymerase processivity
44/62 ATPase and 45 enhance the processivity of T4 DNA polymerase 43 Continuous ATP hydrolysis by 44/62 is not required for enhanced processivity Discovered that polymerase was not very processive on its own, but other proteins that could hydrolyze ATP could enhance this processivity Was this ATP hydrolysis needed continuously throughout the polymerization or just at the beginning? Execution Point, when is an activity no longer needed? Requires the ability to stage the reaction into different steps and to inactivate an activity after a certain step. IN this case they uses low temperature to slow the polymerizatio and a hairpin to stall it separating the reaction into two steps Then they took advantage of ATPgammaS to inhibit the ATP hydrolysis carried out by the 44/62 oligomer after initial polymerization Polymerase remained processive, so once ATP hydrolysis occurs the factor occur like a sliding clamp to keep the polymerase bound to DNA How do you reconcile tight binding with mobility? Once ATP is hydrolyzed, processivity factors act like a “sliding clamp” for the polymerase and provide processivity without need for further ATP hydrolysis

The sliding clamp is a ring that tethers the polymerase
How a sliding clamp works was immediately suggested by x-ray crystallography, which revealed a donut shaped complex These clamps form a ring around duplex DNA with one side bindng to the DNA polymerase, tethering it to the primer-template Of course to get this ring around the DNA there needs to be a way to open the ring And the processivity factor that hydrolyzes ATP was discovered to be a clamp loader that opens that clamp, brings it to a primer-template junction, and releases the clamp, allowing it to close around the duplex part of the junction.

P I1 I2 I3 I4………….. In A1 A2 A3 A4 An+1 S = substrate S P P = product I = intermediate A = activity How to detect and identify intermediates? How to structurally characterize intermediates? Now as we identify a profusion of activities involved in a mechanism it becomes obvious that these activities must act with the proper order and timing. And method by which this order and timing is established is revealed as one uncovers the dependencies of each activity. That’s because nature often designs an activity to depend on key structural features of the intermediate it acts on. One example of how these carefully crafted dependencies ensure proper order and timing can be seen in the appendix discussion of how the clamp loader works Another example is how all the activities for lagging strand synthesis are coordinated as shown in the next slide. How to identify the proteins/nucleic acids responsible for the activities? How is proper order and timing of activities maintained?

Coordinating DNA Synthesis on the Lagging Strand
Primase synthesizes primer Clamp-loader positions clamp around primer-template Polymerase loads onto primer-template and binds to clamp Primase synthesizes primer for next okazaki fragment Here we see many events that must occur, some sequentially and others concurrently We start with the need to synthesize a primer for each okazaki fragment. The synthesis of the primer, triggers the sliding clamp to load a clamp through multiple protein-protein interactions that allow a “handoff” of the primer from the primase to the sliding clamp. The loading of the clamp now enhances the ability of the DNA polymerase to load onto the clamped primer template While the DNA polymerase is synthesizing the okazaki fragment, the next primer is synthesized although we still don’t really understand how the replisome knows when and where to synthesize the primer. In this cycle the DNA polymerase processivity must be highly regulated. During okazaki fragment synthesis the processivity must be high. However, once the fragment is completed, the processivity must be lost so that the polymerase can be released to load onto the next primer template junction How the processivity is regulated is still not fully understood. Two major signals have been hypothesized and there is evidence for both: (1) the collision of the polymerase with the 5’ end of the nascent strand in front of it is one proposed signal; (2) the synthesis of the new primer is the other. The clamp that is left behind has been implicated in additional tasks that are not explicitly shown here. Among these are: (1) recruitment of the proteins required for okazaki fragment maturation; (2) recruitment of the mismatch repair machinery that was covered by the previous lecture; (3) recruitment of histone deposition and chromatin assembly proteins for eukaryotes. Finally, after all these additional clamp functions are performed, a clamp unloader some unknown signal must direct the clamp to be released so it can be recycled onto another primer-template junction. Polymerase synthesizes okazaki fragment What regulates where and when primers are made? Polymerase dissociates from clamp to load onto next primer Clamp-loader loads clamp What regulates polymerase processivity? Okazaki fragment maturation is completed Clamp-UNloader eventually releases clamp for reuse on other okazaki fragments What directs when clamps are released? Adapted from Molecular Biology of the Cell. 4th Ed.

Keeping the Lagging Strand Polymerase at the Replication Fork
Processive synthesis of okazaki fragments by lagging strand polymerase suggests tethering to leading strand replication proteins at the fork, generating a dynamic lagging strand loop (trombone model). In E. coli, tau dimer tethers by binding two core polymerases in the Pol III holoenzyme Finally let me end with one higher order processivity issue that has been hinted at but not explicitly stated. We have discussed polymerase processivity at the level of repeated nucleotide incorporation. Tethered to the clamp, the polymerase stays stably at a primer template junction to allow continuous uninterrupted synthesis off that primer. However, the lagging strand polymerase also shows processivity at the level of repeated okazaki fragment synthesis The polymerase may dissociate from the primer template junction after each fragment is completed, but stays stably with the replication fork so that it can be recycled for multiple rounds of okazaki fragment synthesis. Bruce Alberts came up with a very creative structural solution to this higher-order processivity function He hypothesized that the lagging strand polymerase could be tethered to the replication fork through interaction with other stable fork components, such as the helicase or the leading strand polymerase. However to accommodate this tethering, the DNA template had to be redrawn from its usual Y-like structure to a structure with a loop that expanded during synthesis, then collapsed when the lagging strand polymerase, one of the anchors at the base of this loop, was released from the just completed okazaki fragment. As the polymerase reengaged with the next primer template a new loop would be formed. These cycles of loop expansion and contraction reminded Bruce of a trombone, so this new conceptualization of the replication fork was referred to as the trombone model. How does one accumulate evidence for such a new structure behaving in such a dynamic fashion. Initially, protein-protein interactions compatible with polymerase tethering to the fork were identified. For example the PolII holoenzme was found to be a dimer of two core DNA polymerase, and the dimerization protein tau was found to associate with the helicase dnaB Later, EM pictures compatible with the notion of a trombone loop were observed,although not with the resolution to really confirm the model. More recently, single molecule studies looking at the tension and movement of the lagging strand have generated results consistent with the dynamic expansion and sudden collapse of a loop of the appropriate size. [COULD BE DISCUSSION PAPER??] Thus, there is now reasonable evidence for a model that started simply as a creative concept in Bruce’s imagination. B clamp core Complex clamp-loader t dimer B clamp core Pol III holoenzyme Predicted lagging strand “loop” seen in EM; dynamic loop behavior detected by single molecule analysis Figures from Molecular Biology of the Cell. 4th Ed.

Trombone Model from Cell Snapshots (Cell 141:1088)
How many polymerases can interact with each clamp? How do primase and helicase interact yet work in opposite directions? What holds leading and lagging strand polymerases together in other systems? This slide reviews the trombone model in slow motion along with a few of the many questions that still remain about how the replisome at the fork works One can also see an animated rendition of the model on the website. Are leading and lagging polymerization coordinated? See Movie at

Replication forks must deal with many problems and dangers
Spontaneous genomic insults are now thought to originate from replication accidents DNA lesions induce responses to: (1) protect stalled forks (2) bypass lesions (3) delay further initiation (4) block cell cycle 2 We have discussed the replisome at the replication fork as if it were a train on an open smooth track, and once you get the train going it is a smooth ride to the end. However, in reality those tracks have many obstacles and defects and there is much active research trying to understand how the replication machinery deals with them. Obstacles include DNA binding protein, higher order chromatin, and active transcription Defects include DNA lesions that can stall forks and require the cell to figure out how to get through them without introducing a mutation or rearrangment in the genome. As shown in the figure, the cell has a mechanism to sense these lesions and then to institute several protective responses that are the source of much active investigation. First it must protect the stalled fork and prevent it from disassembling or collapsing’ Second if the lesion can’t be fixed immediately it must find a way of replicating past it. This can involve recombinational tricks as well as the translesion polymerases I discussed earlier Third, it delays initiation of origins that haven’t fired yet so that other lesions can be fixed Finally, it must signal for a delay in mitosis so the problem can be resolved and replication completed before mitosis commences Failure to execute these responses properly is thought to be a source of genome instability that can contribute to cancer and genetic disorders. 3 1 4 Segurado & Tercero, Biol. Cell (2009) 11:

Appendix Bioreg 2015 Replication Lecture 2

Full interpretation of the Cairns theta structure
Label E. coli ~ 2 generations with radioactive thymidine (H ) 3 fork Tt TT daughter parent D Gently lyse cells and let DNA settle and stick onto a membrane Autoradiograph with coating of photographic emulsion Develop emulsion and analyze DNA structures under microscope, quantifying lengths Infer double-strand labeling (TT) vs single-strand labeling (Tt) from quantification of silver grain density At the time label was added the great grandparent molecule, which had initiated from an origin near the bottom left corner, had replicated all but the region from C to D (marked by arrowheads). As this round of replication was completed the resulting grandparent molecule became labeled on one strand just between C and D around the termination zone for replication. Initiation and completion of the next round of replication generated the parent molecule with one strand fully labeled and the other (inherited from the grandparent molecule) labeled only from C to D. Thus, the parental molecule was labeled on both strands between C and D before it initiated the second round of replication. This parent molecule was then caught in the act of replicating with two thirds of it replicated by forks X and Y, generating two daughter arms labeled A and B. Arm A was derived from the mostly unlabeled parental strand and is thus mostly labeled only on the new daughter strand (except from D to X). Arm B was derived from the labeled parental strand and is thus labeled on both strands. The unreplicated parental segment contains much of the double labeling of the termination zone between X and C plus a portion (C to Y) that is single strand labeled

Summary of Activities and Proteins at the Replication Fork
Note: Many of these activities are also required for DNA repair or recombination, and in several cases the same proteins are used Diagram shows prokaryotic 5’>3’ helicase on lagging strand 3’>5’ eukaryotic helicase would be placed on leading strand Task Activity E. coli Eukaryotes unwind parental strands helicase DnaB Mcm2-7, Cdc45, GINS prime DNA synthesis primase primase DNA Pol a-primase stabilize SS DNA SSBP SSBP RPA1-3 * ** synthesize DNA polymerase DNA Pol III core DNA Pol e, DNA Pol d ensure processivity clamp loader, clamp g-complex, b subunit RFC1-5, PCNA So as a summary here are the replication tasks required at the fork, the activities that perform these tasks, and the specific proteins that provide these activities in Prokaryotes and Eukaryotes. Just as all the activities of the clamp loader must be coordinated, there must be a larger coordination between all the activities at the fork that make sure these activities don’t interfere with each other but act in the proper order. So after taking apart the replication machine to it component parts and activities, the next challenge is to figure out how nature has assembled them into an ensemble whose coordinated function is greater than the sum of its parts. A lot of effort is now being made to understand this coordination by figuring out how these activities are inhibited and/or activated depending on the status of the DNA and the other proteins. coord leading and lagging ? t subunit Ctf4? unlink parental strands topoisomerase Topo I/Gyrase, Topo IV Topo I/Topo II replace primer polymerase/nuclease DNA Pol I/RNaseH DNA Pol d, FenI, Dna2 connect okazaki fragments ligase DNA Ligase DNA Ligase I * DNA Pol III Holoenzyme **  leading,  lagging

Key Interactions Order Activities
E. Coli Clamp-Loader (g dd’) loads the Clamp (b ) onto DNA through the ordered execution of activities, each of which is dependent on the intermediate generated by the previous activity 3 2 Key Interactions Order Activities Clamp Loading Model d alone can bind and open clamp interface d’ binds d and blocks interaction with clamp (sequesters d in the clamp-loader) g has ATPase activity ATP binding induces conformational change in g and releases d from d’ (allows g to bind and open clamp) A protein ring topological linked to the DNA is a great solution for a sliding clamp but it poses a new problem. How do you open the ring, bring it to a primer template junction in the right orientation, and then close the ring around the junction. Later, when it has done its many jobs (which includes recruiting and coordination proteins long after the DNA polymerase is gone), the ring has to be removed This loading and unloading of the clamp is performed by a protein machine called a clamp loader. Protein machines can perform a complex series of biochemical steps. This requires that the steps be performed in the correct order on specific substrates, so specificity and order are critical. To do this protein machines have many different activities but not all of them can be active at the same time. Each activity has dependencies that help to enforce the specificity and order of events In many cases the intermediate generated by one biochemical event may be necessary for unleashing the activity of a subsequent biochemical event. For example, the clamp loader doesn’t bind well to primer template junctions until it binds to the clamp. It can’t bind the clamp until it has undergone a conformational change that exposes a domain that can bind the clamp It can’t undergo the conformational change until it first bind to ATP What biochemist do to dissect such a machine is to define all its activities and determine their dependencies, I.e. the conditions required for optimal activity Then they fit those dependencies into the logic of the task the machine needs to do to come up with an order of events. Clamp binding inhibits g ATPase (prevents premature clamp release) Clamp binding enhances clamp-loader binding to primer-template ( promotes clamp delivery to DNA) Primer-template binding stimulates g ATPase (allows g to release and close clamp to complete loading) Energetics Clamp opening depends on protein-ATP (g - ATP) and protein-protein (b - d) binding energies Clamp closing depends on ATP hydrolysis

The Replication Fork and Replisome

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