Chapter 9- DNA Replication

DNA polymerases

- A class of enzymes that catalyzes the synthesis of DNA

Loaded helicases are activated by two protein kinases:

- CDK (cyclin dependent kinase) - DDK (Dbf4-dependent kinase)

the frequency of primase function on the two strands is dramatically different

- Each leading strand requires only a single RNA primer. In contrast, the discontinuous synthesis of the lagging strand means that new primers are needed for each Okazaki fragment. - Because a single replication fork can add hundreds of thousands of nucleotides to a primer, synthesis of the lagging strand can require hundreds of Okazaki fragments and their associated RNA primers

Helicase Activation Alters Interactions

- The helicase is initially loaded around dsDNA as a head-to-head dimer, at the replication fork it is thought to act as a single Mcm2-7 hexamer encircling ssDNA. - Thus, during the activation events, one strand of DNA must be ejected from the central channel of each helicase, and the interactions between the two Mcm2-7 complexes must be disrupted

For the synthesis of DNA to proceed two key substrates must be present

1- new synthesis requires the four deoxynucleoside triphosphates—dGTP, dCTP, dATP, and dTTP. 2- a particular arrangement of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) called a primer: template junction

Nucleotides represent an important class of drugs used to treat cancer and viral infections

Nucleotides that meet some but not all of the requirements for use by DNA polymerase can inhibit DNA synthesis by terminating elongation

The relatively weak interaction between the E. coli primase and DNA helicase is important for regulating the length of Okazaki fragments

- A tighter association would result in more frequent primer synthesis on the lagging strand and therefore shorter Okazaki fragments. - Similarly, aweaker interaction would result in longer Okazaki fragments.

These ring-shaped protein complexes encircle one of the two single strands at the replication fork adjacent to the single-stranded:double-stranded junction.

- DNA helicases act processively - Each time they associate with substrate, they unwind multiple base pairs of DNA. The ringshaped hexameric DNA helicases found at replication forks exhibit high processivity because they encircle the DNA. - Release of the helicase from its DNA substrate therefore requires the opening of the hexameric protein ring, which is a rare event. - Alternatively, the helicase can dissociate when it reaches the end of the DNA strand that it has encircled

High processivity at the replication fork ensures rapid chromosome duplication

- DNA polymerases at the replication fork synthesize thousands to millions of base pairs without releasing from the template. - Despite this, when looked at in the absence of other proteins, the DNA polymerases that act at the replication fork are only able to synthesize 20-100 bp before releasing from the template

The chemistry of DNA synthesis requires that the new chain grows by extending the 30 end of the primer

- Feature of the synthesis of both RNA and DNA. - The phosphodiester bond is formed in an SN2 reaction in which the hydroxyl group at the 3~ end of the primer strand attacks the alpha-phosphoryl group of the incoming nucleoside triphosphate. - The leaving group for the reaction is pyrophosphate, which is composed of the beta-phosphate and gamma-phosphate of the nucleotide substrate.

The events of eukaryotic replication initiation occur at distinct times in the cell cycle

- Helicase loading occurs at all replicators during G1 (before S phase). - Replicator or origin activation, including helicase activation and replisome assembly, only occurs after cells enter S phase

kinetic proofreading

- In which an enzyme favors catalysis using one of several possible substrates by dramatically increasing the rate of bond formation only when the correct substrate is present

How can the activity of a DNA polymerase be measured?

- Incorporation Assays Can Be Used to Measure Nucleic Acid and Protein Synthesis - measures the incorporation of labeled dNTP precursors into DNA molecules - dNTPs are labeled by including radioactive atoms in a part of the nucleotide that will be retained in the final DNA product (e.g., by replacing the phosphorous atom in the a-phosphate with the radioactive isotope 32P) - Alternatively, nucleotides can be synthesized with fluorescent molecules in the place of the methyl group on dTTP - This methyl group is not involved in base pairing, and DNA polymerases can readily accommodate much larger moieties in this location. - these modifications allow easy monitoring of the labeled nucleotide using film or sensitive photomultipliers to detect emitted electrons or photons

DNA polymerases is different than other enzymes

- Other enzymes have one active site that catalyzes one reaction - DNA polymerase uses a single active site to catalyze the addition of any of the four deoxynucleoside triphosphates. - It accomplishes this catalytic flexibility by exploiting the nearly identical geometry of the A:T and G:C base pairs

To complete DNA replication, the RNA primers used for initiation must be removed and replaced with DNA

- Removal of the RNA primers can be thought of as a DNA-repair event, - this process shares many of the properties of excision DNA repair

The Identification of Origins of Replication and Replicators

- Replicator sequences are typically identified using genetic assays. - the first yeast replicators were identified using a DNA transformation assay

The atomic structure of various DNA polymerases bound to primer:template junctions

- Reveal that the DNA substrate sits in a large cleft that resembles a partially closed right hand. - Based on the hand analogy, the three domains of the polymerase are called the thumb, fingers, and palm.

Only a subset of the proteins that assemble at the origin goes on to function as part of the eukaryotic replisome

- The CMG complex and the three DNA polymerases become part of the replication fork machinery - other factors are only required to assemble the replication fork proteins (such as Cdc6 and Cdt1) and are released or destroyed after their role is complete

These alternate forms of the bases permit incorrect base pairs to be correctly positioned for catalysis

- When the nucleotide returns to its "correct" state, the incorporated nucleotide is mismatched with the template and must be eliminated. - Removal of these incorrectly base-paired nucleotides is mediated by a type of nuclease that was originally identified in the same polypeptide as the DNA polymerase.

CDKs (cyclin-dependent kinases)

- the regulation is tightly coupled to the function of CDKs - These enzymes play seemingly contradictory roles in regulating replication

The palm domain job

- the thumb interacts with the DNA that has been most recently synthesized. serves two purposes 1- it maintains the correct position of the primer and the active site. 2- the thumb helps to maintain a strong association between the DNA polymerase and its substrate. - This association contributes to the ability of the DNA polymerase to add many dNTPs each time it binds a primer: template junction

Step 1 : The removal of mismatched nucleotides is facilitated by the reduced ability of DNA polymerase to add a nucleotide adjacent to an incorrectly base-paired primer

- Mispaired DNA alters the geometry between the 3'-OH and the incoming nucleotide because of poor interactions with the palm region. - This altered geometry reduces the rate of nucleotide addition in much the same way that addition of an incorrectly paired dNTP reduces catalysis - when a mismatched nucleotide is added, it both decreases the rate of new nucleotide addition and increases the rate of proofreading exonuclease activity.

Processivity is facilitated by sliding of DNA polymerases along the DNA template.

- Once bound to a primer:template junction, DNA polymerase interacts tightly with much of the double-stranded portion of the DNA in a sequence-nonspecific manner. - These interactions include electrostatic interactions between the phosphate backbone and the thumb domain and interactions between the minor groove of the DNA and the palm domain. - The sequence-independent nature of these interactions permits the easy movement of the DNA even after it binds to polymerase. - Each time a nucleotide is added to the primer strand, the DNA partially releases from the polymerase. (The hydrogen bonds with the minor groove are broken, but the electrostatic interactions with the thumb are maintained.) - The DNA then rapidly rebinds to the polymerase in a position that is shifted by 1 bp using the same sequence-nonspecific mechanism.

Step 2: When a mismatched base pair is present in the polymerase active site, the primer:template junction is destabilized, creating several base pairs of unpaired DNA.

- The DNA polymerase active site binds such a mismatched template poorly, but the exonuclease active site has a 10-fold higher affinity for single-stranded 3' ends. - the newly unpaired 30 end moves from the polymerase active site to the exonuclease active site. The incorrect nucleotide is removed by the exonuclease (an additional nucleotide may also be removed).

polymerase switching

- The process of replacing DNA Pol a/primase with DNA Pol d or Pol 1 - Because of its relatively low processivity DNA Pol a/primase is rapidly replaced by the highly processive DNA Pol d and Pol 1. - results in three different DNA polymerases functioning at the eukaryotic replication fork. - DNA Pol d and 1 are specialized to synthesize different strands at the replication fork, with DNA Pol 1 synthesizing the leading strand and DNA Pol d the lagging strand. - As in bacterial cells, the majority of the remaining eukaryotic DNA polymerases are involved in DNA repair.

polarity of the DNA helicase

- The property where each DNA helicase moves along ssDNA in a defined direction - DNA helicases can have a polarity of either 5'!3' or 3'!5'. This direction is always defined according to the strand of DNA bound (or encircled for a ring-shaped helicase), rather than the strand that is displaced. - In the case of a DNA helicase that functions on the lagging-strand template of the replication fork, the polarity is 5'!3' to allow the DNA helicase to proceed toward the duplex region of the replication fork. As is true for all enzymes that move along DNA in a directional manner, movement of the helicase along ssDNA requires the input of chemical energy. For helicases, this energy is provided by ATP hydrolysis.

- the initiator protein is the only sequence-specific DNA binding protein involved in the initiation of replication

- The remaining proteins required for replication initiation do not bind to a DNA sequence specifically. - Instead, these proteins are recruited to the replicator through a combination of protein-protein interactions and affinity for specific DNA structures (e.g., ssDNA or a primer: template junction). - Indeed, for many eukaryotic cells even the initiator protein does not show sequencespecific DNA-binding activity.

Okazaki fragments

- The resulting short fragments of new DNA formed on the lagging strand - vary in length from 1000 to 2000 nucleotides in bacteria and from 100 to 400 nucleotides in eukaryotes. - Shortly after being synthesized, Okazaki fragments are covalently joined together to generate a continuous, intact strand of new DNA - Okazaki fragments are therefore transient intermediates in DNA replication

The proteins that act at the replication fork interact tightly but in a sequence-independent manner with the DNA

- both DNA helicase and topoisomerase perform their functions without permanently altering the chemical structure of DNA or synthesizing any new molecule. - DNA helicase breaks only the hydrogen bonds that hold the two strands of DNA together without breaking any covalent bonds. - Although topoisomerases break one or two of the targeted DNA's covalent bonds, each bond broken is precisely re-formed before the topoisomerase releases the DNA. - Instead of altering the chemical structure of DNA, the action of these enzymes results in a DNA molecule with an altered conformation. - Importantly, these conformational alterations are essential for the duplication of the large dsDNA molecules that are the foundation of both bacterial and eukaryotic chromosomes.

DNA helicases

- enzyme that catalyze the separation of the two strands of duplex DNA. These enzymes bind to and move directionally along ssDNA using the energy of nucleoside triphosphate (usually ATP) binding and hydrolysis to displace any DNA strand that is annealed to the bound ssDNA. - Typically, DNA helicases that act at replication forks are hexameric proteins that assume the shape of a ring

Once the initiator binds to the replicator

- the remaining steps in the initiation of replication are largely driven by protein-protein interactions and protein-DNA interactions that are sequence-independent. - The end result is the assembly of two replisomes

Organisms solve the end replication problem in a variety of ways

1 - use a protein instead of an RNA as the primer for the last Okazaki fragment at each end of the chromosome 2 - Telomeres' unique structure acts as a novel origin of replication that compensates for the end replication problem. This origin does not interact with the same proteins as other eukaryotic origins, but it instead recruits a specialized DNA polymerase called telomerase

How is this regulation achieved?

1- CDK are required to activate loaded helicases to initiate DNA replication. 2- CDK activity inhibits helicase loading - these different roles allow one enzyme to control the oscillation between the two states of replication initiation. - CDK levels are low during G1, allowing helicase loading but preventing helicase activation. - Entry into the S phase of the cell cycle is coupled with a rapid increase in CDK activity, driving activation of loaded helicases but simultaneously preventing new helicase loading. - CDK levels remain elevated during the remainder of the cell cycle (S, G2, and M phases).

The Driving Force for DNA Synthesis

1- The free energy from the addition of a nucleotide to a growing polynucleotide chain of length n - It is rather small (delta G = -3.5 kcal/mol). 2- Additional free energy is provided by the rapid hydrolysis of the pyrophosphate into two phosphate groups by an enzyme known as pyrophosphatase - The net result of nucleotide addition and pyrophosphate hydrolysis is the breaking of two high-energy phosphate bonds. - Therefore,DNA synthesis is a coupled process. - This is a highly favorable reaction with a delta G of -7 kcal/mol - This corresponds to an equilibrium constant (Keq) of ~10^5 - high Keq means that the DNA synthesis reaction is effectively irreversible

How is the processivity of these enzymes increased so dramatically at the replication fork?

1- association with proteins called sliding DNA clamps - In the absence of the sliding clamp, a DNA polymerase dissociates and diffuses away from the template DNA on average once every 20-100 bp synthesized. - In the presence of the sliding clamp, the DNA polymerase still disengages its active site from the 3'-OH end of the DNA frequently, but the association with the sliding clamp prevents the polymerase from diffusing away from the DNA - By keeping the DNA polymerase in close proximity to the DNA, the sliding clamp ensures that the DNA polymerase rapidly rebinds the same primer:template junction, vastly increasing the processivity of the DNA polymerase.

The fingers domain job

1- catalysis. - Several residues located within the fingers bind to the incoming dNTP - once a correct base pair is formed between the incoming dNTP and the template, the finger domain moves to enclose the dNTP - This closed form of the polymerase "hand" stimulates catalysis by moving the incoming nucleotide into close contact with the catalytic metal ions 2- associates with the template region, leading to a nearly 90 degrees turn of the phosphodiester backbone between the first andsecond bases of the template. -This bend serves to expose only the first template base after the primer at the catalytic site and avoids any confusion concerning which template base should pair with the next nucleotide to be added

Eukaryotic helicase loading requires four separate proteins to act at each replicator

1- the recognition of the replicator by the eukaryotic initiator, ORC, bound to ATP - cells enter the G1 phase of the cell cycle, ORC bound to the origin recruits two helicase loading proteins (Cdc6 and Cdt1) and two copies of the Mcm2-7 helicase to the origin

The DNA sequences of known replicators share two common features

1- they include a binding site for the initiator protein that nucleates the assembly of the replication initiation machinery. 2- they include a stretch of AT rich DNA that unwinds readily but not spontaneously. - Unwinding of DNA at replicators is controlled by the replication initiation proteins, and the action of these proteins is tightly regulated in most organisms

replicon

All of the DNA replicated from a particular origin of replication - the single chromosome found in E. coli cells has only one origin of replication, the entire chromosome is a single replicon. - In contrast, the presence of multiple origins of replication divides each eukaryotic chromosome into multiple replicons—one for each origin of replication - The replicon model proposed two components that controlled the initiation of replication: the replicator and the initiator

Each of theeukaryotic DNA polymerases DNA Pol d, DNA Pol 1, and DNA Pol a/primase is composed of multiple subunits

DNA Pol a/primase is specifically involved in initiating new DNA strands. - This four-subunit protein complex consists of a two-subunit DNA Pol a and a two-subunit primase. - After the primase synthesizes an RNA primer, the resulting RNA primer:template junction is immediately handed off to the associated DNA Pol a to initiate DNA synthesis.

Primer: template junction job

Formally, - only the primer portion of the primer:template junction is a substrate for DNA synthesis because only the primer is chemically modified during DNA synthesis. - The template provides only the information necessary to select which nucleotides are added. Nevertheless, both a primer and a template are essential for all DNA synthesis

What does DNA polymerase monitor

The DNA polymerase monitors the ability of the incoming nucleotide to form an A:T or G:C base pair, rather than detecting the exact nucleotide that enters the active site - Only when a correct base pair is formed are the 3~-OH of the primer and the a-phosphate of the incoming nucleoside triphosphate in the optimum position for catalysis to occur. - Incorrect base pairing leads to dramatically lower rates of nucleotide addition as a result of a catalytically unfavorable alignment of these substrates

How do eukaryotic cells control the activity of hundreds or even thousands of origins of replication such that not even one is activated more than once during a cell cycle?

The answer lies in the oscillation between two replication states that occurs once per cell cycle. - During G1, cells are in the helicase loading phase and are competent for helicase loading but unable to activate the loaded helicases. - Upon entry into S phase and continuing throughout G2 and Mphase, helicases loaded during G1 can be activated, but new helicase loading is strictly inhibited - Importantly, the conditions for helicase loading and activation are incompatible with one another

replication fork

The junction between the newly separated template strands and the unreplicated duplex DNA

the end replication problem

The requirement for an RNA primer to initiate all new DNA synthesis creates a dilemma for the replication of the ends of linear chromosomes

replisome

Thecombination of all of the proteinsthat functionat the replication fork is referred to as the replisome - these proteins form a finely tuned factory for DNA synthesis that contains multiple interacting machines. - Individually, these machines perform important specific functions. - When brought together, their activities are coordinated by the interactions between them

Processivity

a characteristic of enzymes that operate on polymeric substrates

The replication fork moves

continuously toward the duplex region of unreplicated DNA, leaving in its wake two ssDNA templates that each direct the synthesis of a complementary DNA strand

proofreading exonuclease

enzymes degrade DNA starting from a 3~ DNA end (i.e., from the growing end of the new DNA strand).

exonucleases vs endonucleases

exonucleases: that can only degrade from a DNA endonucleases: that can cut within a DNA strand

Degree of processivity

in the case of DNA polymerase it is defined as the average number of nucleotides added each time the enzyme binds a primer:template junction.

The antiparallel nature of DNA creates a complication for the simultaneous replication of the two exposed templates at the replication fork.

leading strand: The newly synthesized DNA strand directed by the template replicated continuously as the replication fork moves. by elongating a 3' end lagging strand: The new DNA strand directed by the template directing the DNA polymerase to move in the opposite direction of the replication fork.

telomeres

the ends of eukaryotic chromosomes, and they are generally composed of head-to-tail repeats of a TG-rich DNA sequence.

Antiparallel Elongation

The antiparallel structure of the double helix affects replication

sliding clamp loaders

- A special class of protein complexes, catalyze the opening and placement of sliding clamps on the DNA. - These enzymes couple ATP binding and hydrolysis to the placement of the sliding clamp around primer: template junctions on the DNA - The clamp loader also removes sliding clamps from the DNA when they are no longer in use, although this does not require ATP hydrolysis. Like DNA helicases and topoisomerases, these enzymes alter the conformation of their target (the sliding clamp) but not its chemical composition.

approximately one mistake in every 1010 bp added

- A system based only on base-pair geometry and the complementarity between the bases cannot reach the extraordinarily high levels of accuracy that are observed for DNA synthesis in the cell. - A major limit to DNA polymerase accuracy is the occasional (about one in 105 times) flickering of the bases into the "wrong" tautomeric form (imino or enol)

DNA polymerases show an impressive ability to distinguish between ribonucleoside and deoxyribonucleoside triphosphates (rNTPs and dNTPs)

- Although rNTPs are present at approximately 10-fold higher concentration in the cell, they are incorporated at a rate that is more than 1000-fold lower than dNTPs - This discrimination is mediated by the steric exclusion of rNTPs from the DNA polymerase active site - In DNA polymerase, the nucleotide-binding pocket cannot accommodate a 2~-OH on the in-coming nucleotide. - This space is occupied by two amino acids that make van der Waals contacts with the sugar ring. Changing these amino acids to other amino acids with smaller side chains results in a DNA polymerase with significantly reduced discrimination between dNTPs and rNTPs.

the events required for eukaryotic cell division occur at distinct times during the cell cycle

- Chromosomal DNA replication occurs only during the S phase of the cell cycle. - During this time, all of the DNA in the cell must be duplicated exactly once. - Incomplete replication of any part of a chromosome causes inappropriate links between daughter chromosomes. - Segregation of linked chromosomes causes chromosome breakage or loss - Re-replication of even limited amounts of eukaryotic DNA leads to DNA lesions that are difficult for the cell to repair. - the temporal separation of helicase loading from helicase activation and replisome assembly during the eukaryotic cell cycle ensures that each chromosome is replicated only once during each cell cycle

The central role of DNA polymerases in the efficient and accurate replication of the genome has resulted in the evolution of multiple specialized DNA polymerases. in E. Coli

- DNA polymerase III: (DNA Pol III) is the primary enzyme involved in the replication of the chromosome. must be highly processive - DNA Pol III holoenzyme: DNA Pol III is generally found to be part of a larger complex that confers very high processivity - DNA polymerase I: specialized for the removal of the RNA primers that are used to initiate DNA synthesis. has a 5' exonuclease that allows it to remove RNA or DNA immediately upstream of the site of DNA synthesis. not highly processive, adding only 20-100 nucleotides per binding event - Because both DNA Pol I and DNA Pol III are involved in DNA replication, both of these enzymes must be highly accurate. Thus, both proteins include an associated proofreading exonuclease

Replication Fork Enzymes Extend the Range of DNA Polymerase Substrates

- DNA polymerase can only efficiently extend 3'-OH primers annealed to ssDNA templates. - The addition of primase, DNA helicase, and topoisomerase dramatically extends the possible substrates for DNA polymerase. - Primase provides the ability to initiate new DNA strands on any piece of ssDNA. Of course, the use of primase also imposes a requirement for the removal of the RNA primers to complete replication. - Similarly, strand separation by DNA helicase and dissipation of positive supercoils by topoisomerase allow DNA polymerase to replicate dsDNA.

Removal of the RNA primer leaves a gap in the dsDNA that is an ideal substrate for DNA polymerase—a primer:template junction

- DNA polymerase fills this gap until every nucleotide is base-paired, leaving a DNA molecule that is complete except for a break in the phosphodiester backbone between the 3'-OH and 5'-phosphate of the repaired strand - This "nick" in the DNA can be repaired by an enzyme called DNA ligase - DNA ligases use high-energy co-factors (such as ATP) to create a phosphodiester bond between an adjacent 5'-phosphate and 3'-OH. Only after all RNA primers are replaced by DNA and the associated nicks are sealed is DNA synthesis complete.

Catalysis by DNA polymerase is rapid

- DNA polymerases are capable of adding as many as 1000 nucleotides /sec to a primer strand - The rate of DNA synthesis is dramatically increased by adding multiple nucleotides per binding event - It is the initial binding of polymerase to the primer:template junction that is rate-limiting for DNA synthesis - In a typical DNA polymerase reaction, it takes ~1 sec for the DNA polymerase to locate and bind a primer:template junction - Once bound, addition of a nucleotide is very fast (in the millisecond range). Thus, a completely nonprocessive DNA polymerase would add ~1 bp/sec. - In contrast, the fastest DNA polymerases add as many as 1000 nucleotides/sec by remaining associated with the template for thousands of rounds of dNTP addition - Consequently, a highly processive polymerase increases the overall rate of DNA synthesis by as much as 1000-fold compared with a nonprocessive enzyme

Origin of recognition complex (ORC)

- In eukaryotic cells, the initiator is a six-protein complex. - ORC recognizes a conserved sequence found in yeast replicators, called the "A element," as well as a second, less-conserved B1 element - Like DnaA, ORC binds and hydrolyzes ATP. - ATP binding is required for sequence-specific - DNA binding at the origin, and ATP hydrolysis is required for ORC to participate in the loading of the eukaryotic DNA helicase onto the replicator DNA - Unlike DnaA, binding of ORC to yeast replicators does not lead to strand separation of the adjacent DNA. - Nevertheless, ORC is required to recruit, either directly or indirectly, all of the remaining replication proteins to the replicator

Once released from a DNA polymerase, sliding clamps are not immediately removed from the replicated DNA

- Instead, other proteins that function at the site of recent DNA synthesis interact with the clamp proteins. - enzymes that assemble chromatin in eukaryotic cells are recruited to the sites of DNA replication by an interaction with the eukaryotic sliding DNA clamp (called "PCNA"). - eukaryotic proteins involved in Okazaki fragment repair also interact with sliding clamp proteins. - In each case, by interacting with sliding clamps, these proteins accumulate at sites of new DNA synthesis where they are needed most. - Sliding clamp proteins are a conserved part of the DNA replication apparatus derived from organisms as diverse as viruses, bacteria, yeast, and humans. Consistent with their conserved function, the structure of sliding clamps derived from these different organisms is also conserved

this arrangement of enzyme and DNA poses problems for the binding of the DNA helicase to the DNA substrate in the first place

- It is most obvious for circular chromosomes, where there is no DNA end for the DNA helicase to thread onto - because helicases are almost always loaded onto the DNA at internal sites of linear chromosomes, the same problem exists during the replication of these DNAs. - there are specialized mechanisms that open the DNA helicase ring and place it around the DNA before re-forming the ring. - This topological linkage between proteins involved in DNA replication and their DNA substrates is a common mechanism to increase processivity

an ordered series of events occurs each time the DNA polymerase adds a nucleotide to the growing DNA chain

- The incoming nucleotide base-pairs with the next available template base. - - This interaction causes the fingers of the polymerase to close around the base-paired dNTP. - This conformation of the enzyme places the critical catalytic metal ions in a position to catalyze formation of the next phosphodiester bond. - Attachment of the base-paired nucleotide to the primer leads to the reopening of the fingers and the movement of the primer:template junction by one base pair. - The polymerase is then ready for the next cycle of addition. Importantly, each of these events is strongly stimulated by correct base pairing between the incoming dNTP and the template

The 5' exonuclease of DNA Pol I can remove the RNA-DNA linkage that is resistant to RNase H

- The lowprocessivity of DNA Pol I readily synthesizes across the short region previously occupied by an RNA primer ( < 10 nucleotides) but is released before degrading and resynthesizing large amounts of DNA that was primed by the RNA. - Finally, when DNA Pol I completes its function, only a nick is present in the DNA - The remaining three DNA polymerases in E. coli are specialized for DNA repair and lack proofreading activities.

primer: template junction

- The primer: template junction has two key components. - The template provides the ssDNA that directs the addition of each complementary deoxynucleotide. T- he primer is complementary to, but shorter than, the template. - The primer must have an exposed 30-OH adjacent to the single-strand region of the template. - It is this 30-OH that will be extended by nucleotide addition

initiator protein

- The second component of the replicon model - specifically recognizes a DNA element in the replicator and activates the initiation of replication - All initiator proteins select the sites that will become origins of replication - all of the known initiator proteins are regulated by ATP binding and hydrolysis and share a common core AAA^+ ATP-binding motif related to, but distinct from, that used by sliding DNA clamp loaders.

The template strand for DNA synthesis has the opposite orientation of the growing DNA strand

- The template strand directs which of the four nucleoside triphosphates is added. - The nucleoside triphosphate that base-pairs with the template strand is highly favored for addition to the primer strand. - Recall that the two strands of the double helix have an antiparallel orientation

sliding DNA clamps

- These proteins are composed of multiple identical subunits that assemble in the shape of a "doughnut." - The hole in the center of the clamp is large enough to encircle the DNA double helix and leave room for a layer of one or two water molecules between the DNA and the protein - These properties allow the clamp proteins to slide along the DNA without dissociating from it. - Importantly, sliding DNA clamps also bind tightly to DNA polymerases bound to primer:template junctions - The resulting complex between the polymerase and the sliding clamp moves efficiently along the DNA template during DNA synthesis

Nucleoside triphosphates have three phosphoryl groups

- They are attached via the 5'-hydroxyl of the 2'-deoxyribose. -The phosphoryl group proximal to the deoxyribose is called the alpha-phosphate - the middle and distal groups are called the beta-phosphate and the gamma- phosphate, respectively.

At the replication fork, the leading and lagging strands are synthesized simultaneously

- This has the important benefit of limiting the amount of ssDNA present in the cell during DNA replication. - When an ssDNA region of DNA is broken, there is a complete break in the chromosome that is much more difficult to repair than an ssDNA break in a dsDNA region. - Moreover, repair of this type of lesion frequently leads to mutation of the DNA

Once an ssDNA template has directed synthesis of its complementary DNA strand, the DNA polymerase must release from the completed dsDNA and the sliding clamp to act at a new primer:template junction.

- This release is accomplished by a change in the affinity between the DNA polymerase and the sliding clamp that depends on the bound DNA. - DNApolymerase bound to a primer:template junction has a high affinity for the clamp. - In contrast, when a DNA polymerase reaches the end of an ssDNA template (e.g., at the end of an Okazaki fragment), the presence of dsDNA in its active site results in a change in conformation that reduces the polymerase's affinity for the sliding clamp and the DNA. - Thus, when a polymerase completes the replication of a stretch of DNA, it is released from the sliding clamp so that it can act at a new primer: template junction

RNaseH

- To replace the RNA primers with DNA - enzyme that recognizes and removes most of each RNA primer. - This enzyme specifically degrades RNA that is base-paired with DNA (the H in its name stands for "hybrid" in RNA:DNA hybrid). - RNase H removes all of the RNA primer except the ribonucleotide directly linked to the DNA end. - This is because RNaseHcan only cleave bonds between two ribonucleotides. - The final ribonucleotide is removed by a 5' exonuclease that degrades RNA or DNA from their 5' ends

Telomerase

- a remarkable enzyme that includes multiple protein subunits and an RNA component - Like all other DNA polymerases, telomerase acts to extend the 3' end of its DNA substrate. - But unlike most DNA polymerases, telomerase does not need an exogenous DNA template

the end replication problem is not observed during the duplication of the leading-strand template, only the lagging strand

- a single internal RNA primer can direct the initiation of a DNA strand that can be extended to the extreme 5' terminus of its template - the requirement for multiple primers to complete lagging-strand synthesis means that a complete copy of its template cannot be made - Even if the end of the last RNA primer for Okazaki fragment synthesis anneals to the final base of the lagging-strand template, - once this RNA molecule is removed, there will remain a short region (the size of the RNA primer) of unreplicated ssDNA at the end of the chromosome

topoisomerases

- act on the unreplicated dsDNA in front of the replication fork - do this by breaking either one or both strands of the DNA without letting go of the DNA and passing the same number of DNA strands through the break. - This action relieves the accumulation of supercoils. - topoisomerases act as a "swivelase" that prevents the accumulation of positive supercoils ahead of the replication fork.

How are new strands of DNA synthesis started?

- all DNA polymerases require a primer with a free 3'-OH. - They cannot initiate a new DNA strand de novo - the cell takes advantage of the ability of RNA polymerases to do what DNA polymerases cannot: start new RNA chains de novo

DNA helicase

- another protein that acts at the replication fork which causes primase activity to be dramatically increased when it associates with it - it unwinds the DNA at the replication fork, creating an ssDNA template that can be acted on by primase - The requirement for an ssDNA template and DNA helicase association ensures that primase is only active at the replication fork.

Why would the DNA polymerase need to degrade the DNA it had just synthesized?

- exonucleases have a strong preference to degrade DNA containing mismatched base pairs - in the rare event that an incorrect nucleotide is added to the primer strand, the exonuclease removes this nucleotide from the 3' end of the primer strand - This "proofreading" of the newly addedDNAgives theDNApolymerase a second chance to add the correct nucleotide.

the first yeast replicators were identified using a DNA transformation assay

- investigators randomly cloned genomic DNA fragments into plasmids lacking a replicator but containing a selectable marker missing in the host cell - the cloned DNA fragment must contain a yeast replicator for the plasmid to be maintained in the host cell after transformation, - The identified DNA fragments were called autonomously replicating sequences (ARSs). - study picture

The palm domain

- is composed of a beta sheet and contains the primary elements of the catalytic site. In particular, this region of DNA polymerase binds two divalent metal ions (typically Mg^2+ or Zn^2+) that alter the chemical environment around the correctly base-paired dNTP and the 3~-OH of the primer

ssDNA-binding proteins

- keep the newly generated ssDNA must remain free of base pairing until it can be used as a template for DNA synthesis - stabilize the separated strands - rapidly bind to the separated strands. Binding of one SSB promotes the binding of another SSB to the immediately adjacent ssDNA

cooperative binding

- occurs because SSB molecules bound to immediately adjacent regions of ssDNA also bind to each other. - This SSB-SSB interaction strongly stabilizes SSB binding to ssDNA and makes sites already occupied by one or more SSB molecules preferred SSB-binding sites. - ensures that ssDNA is rapidly coated by SSB as it emerges from the DNA helicase. (Cooperative binding is a property of many DNA-binding proteins.) - Once coated with SSBs, ssDNA is held in an elongated state that facilitates its use as a template for DNA or RNA primer synthesis. - In contrast to sequence-specific DNA-binding proteins, SSBs make few, if any, hydrogen bonds to the ssDNA bases.

primase does not require an extended DNA sequence to initiate RNA synthesis

- primases prefer to initiate RNA synthesis using an ssDNA template containing a particular trimer (GTA in the case of Escherichia coli primase). - Consistent with this preference, analysis of the E. coli genome sequence shows that the GTA target sequence for E. coli primase is over represented - in the portions of the genome that will be the template for laggingstrand DNA synthesis.

Proteinc kinases

- proteins that covalently attach phosphate groups to target proteins - activated when cells enter S phase - Once activated, DDK targets the loaded helicase, and CDK targets two other replication proteins. - Phosphorylation of these proteins results in the Cdc45 and GINS proteins binding to the Mcm2-7 helicase - Importantly, Cdc45 and GINS strongly stimulate the Mcm2-7 ATPase and helicase activities and together form the Cdc45-Mcm2-7-GINS (CMG) complex, which is the active form of the Mcm 2-7 DNA helicase.

the recognition of the replicator by the eukaryotic initiator, ORC, bound to ATP

- several ORC subunits and the Cdc6 protein are members of the AAAþ family of proteins like DnaC and the subunits of the sliding clamp loaders. Like the sliding clamp loader, ATP binding by ORC and Cdc6 is required for ORC DNA binding and the stable recruitment of the helicase and helicase loading proteins. - ATP hydrolysis by Cdc6 results in the loading of a head-to-head dimer of the Mcm2-7 complex such that they encircle the double-stranded origin DNA. - During this event, Cdt1 and Cdc6 are released from the origin. ORC ATP hydrolysis is thought to reset the process and allowa new round of Mcm2-7 loading to be initiated upon ATP binding to the ORC. - Consistent with the Mcm2-7 complex encircling dsDNA instead of ssDNA, eukaryotic helicase loading does not lead to the immediate unwinding of origin DNA.

genes at the end of the chromosomes would be lost.

- shortening would only occur on one of the two strands of the daughter molecule, after the next round of replication occurs both strands of the daughter molecule would be shorter. - This means that each round of DNA replication would result in the shortening of one of the two daughter DNA molecules. - Obviously, this scenario would disrupt the complete propagation of the genetic material from generation to generation

Primase

- specialized RNA polymerase dedicated to making short RNA primers (5-10 nucleotides long) on an ssDNA template - These primers are subsequently extended by DNA polymerase - Although DNA polymerases incorporate only deoxyribonucleotides into DNA, they can initiate synthesis using either an RNA primer or aDNA primer annealed to the DNA template.

The replicator

- the cis-acting DNA sequences that are sufficient to direct the initiation of DNA replication - the origin of replication is always part of the replicator, sometimes (particularly in eukaryotic cells) the origin of replication is only a fraction of the DNA sequences required to direct the initiation of replication (the replicator).

For a circular chromosome

- the conventional replication fork machinery replicates the entire molecule, but the resulting daughter molecules are topologically linked to each other - After replication of a circular chromosome is complete, the resulting daughter DNA molecules remain linked together as catenanes - To segregate these chromosomes into separate daughter cells, the two circular DNA molecules must be disengaged from each other or "decatenated." This separation is accomplished by the action of type II topoisomerases.

origins of replication

- the physical site on the DNA where the DNA is unwound and DNA synthesis initiates. - The specific sites at which DNA unwinding and initiation of replication occur - Depending on the organism, there may be as few as one or as many as thousands of origins per chromosome

As the strands of DNA are separated at the replication fork, the dsDNA in front of the fork becomes increasingly positively supercoiled

- the result of DNA helicase eliminating the base pairs between the two strands. - If the DNA strands remain unbroken, there can be no reduction in linking number (the number of times the two DNA strands are intertwined) to accommodate this unwinding of the DNA duplex - as the DNA helicase proceeds, the DNA must accommodate the same linking number within a smaller and smaller number of base pairs. - for the DNA in front of the replication fork to remain relaxed, one DNA link must be removed for every `10 bp of DNA unwound. - If there were no mechanism to relieve the accumulation of these supercoils, the replication machinery would grind to a halt in the face of mounting strain placed on the DNA in front of the replication fork.

Telomerase Solves the End Replication Problem by Extending

-When telomerase acts on the 3' end of the telomere, it extends only one of the two strands of the chromosome. - This is accomplished by the lagging-strand DNA replication machinery - By providing an extended 3' end, telomerase provides additional template for the lagging-strand replication machinery - By synthesizing and extending RNA primers using the telomerase extended 3' end as a template, the cell can effectively increase the length of the 5' end of the chromosome as well.

"telomerase RNA" (TER).

1 - The key to telomerase's unusual functions is revealed by the RNA component of the enzyme - This region of the RNA can anneal to the ssDNA at the 3' end of the telomere - Annealing occurs in such away that a part of the RNA template remains single-stranded, creating a primer:template junction that can be acted on by telomerase. 2- one of the protein subunits of telomerase is a member of a class of DNA polymerases that use RNA templates called "reverse transcriptases" (this subunit is called "telomerase reverse transcriptase," or TERT) - Using the associated RNA template, TERT synthesizes DNA to the end of the TER template region but cannot continue to copy the RNA beyond that point

The connections between the components of the DNA Pol III holoenzyme

1 - interactions that occur between the components of the bacterial replication fork. 2 - Additional protein-protein interactions between replication fork proteins facilitate rapid replication fork progression. - The most important of these is an interaction between the DNA helicase (the hexameric dnaB protein) and the DNA Pol III holoenzyme - stimulates the activity of the helicase by increasing the rate of helicase movement 10-fold - DNAhelicase slows down if it becomes separated from the DNA polymerase 3- protein interaction occurs between the DNA helicase and primase. - primase is not tightly associated with the fork - at an interval of about once per second, primase associates with the helicase and SSB-coated ssDNA and synthesizes a new RNA primer.

Job of palm domain

1- role in catalysis - One metal ion reduces the affinity of the 3~-OH for its hydrogen. This generates a 3'O^- that is primed for the nucleophilic attack of the alpha-phosphate of the incoming dNTP. - The second metal ion coordinates the negative charges of the beta-phosphate and gamma-phosphate of the dNTP and stabilizes the pyrophosphate produced by joining the primer and the incoming nucleotide. 2- monitors the base pairing of the most recently added nucleotides - This region of the polymerase makes extensive hydrogen-bond contacts with base pairs in the minor groove of the newly synthesized DNA - These contacts are not base specific but only form if the recently added nucleotides are correctly base-paired. - Mismatched DNA in this region interferes with these minor-groove contacts and dramatically slows catalysis. - The combination of the slowed catalysis and reduced affinity for newly synthesized mismatched DNA allows the release of the primer strand from the polymerase active site, and, in many cases, this strand binds and is acted on by a proofreading nuclease that removes the mismatched DNA

Step 3: The removal of the mismatched base allows the primer:template junction to re-form and rebind the polymerase active site, enabling DNA synthesis to continue

proofreading exonucleases work like a "delete key," removing only the most recent errors. The addition of a proofreading exonuclease greatly increases the accuracy of DNA synthesis. On average, DNA polymerase inserts one incorrect nucleotide for every 10^5 nucleotides added. Proofreading exonucleases decrease the appearance of incorrect base pairs to 1 in every 10^7 nucleotides added