1

Copyright © 2004 by W. H. Freeman & Company

Lehninger Principles of Biochemistry

Fourth Edition

Chapter 25:DNA Metabolism

David L. Nelson and Michael M. Cox

Molecular Biology by Robert F. Weaver4th Edition

DNA metabolism comprises of:Replication – the process by which copies of DNA molecules are faithfully

made;Repair and Recombination – the processes that affect the inherent

structure of the information.

Two very important criteria of DNA replication:Fidelity - DNA replication process requires an exquisite degree of accuracy.

Uncorrected errors that arise during DNA synthesis can have direconsequences, not only because they can permanently affect or eliminate the function of a gene but also because the change is inheritable.

Speed – DNA molecules may contain a million of bases. The machinery (including a myriad of proteins and enzymes) that synthesizes DNA consequently do so with extraordinary speed and fidelity.

Enzymes of DNA metabolism merit careful study for:• understanding the amazing DNA replication process,• increasing importance of these enzymes in medicine,• everyday use of these enzymes in a wide range of modern biochemical

technologies.

Many of the seminal discoveries in DNA metabolism are made with Escherichia coli.

2

Map of the E.coli chromosome The map shows the relative positions of genes encoding many of the proteins important in DNA metabolism.

The number 0 to 100 denote a genetic measurement called minutes. Each minute corresponds to ~40,000 bpalong the DNA molecule of E. coli.

The three-letter names of genes and other elements generally reflect some aspects of their functions.

mut – mutagenesisdna – DNA replicationpol – DNA polymeraserpo – RNA polymeraseuvr – UV resistancerec – recombinationdam – DNA adenine

methylationlig – DNA ligaseTer– termination of replicationori – origin of replication

The number of genes involved in DNA replication provides a hint of

the complexity of this process.

Mysteries:

How organisms create faithful copies of themselves?Results of landmark experiments conducted by the following scientists:

1868 - Friedrich Miescher1944 - Oswald T. Avery, Colin MacLeod, and Maclyn McCarty1952 - Alfred D. Hershey and Martha Chase

proved that DNA is the genetic material.

How the cells produce identical copies of large and complex macromolecules?Speculation – the concept of a template – a structure that would allow molecules to line up in a specific order and get joined, to create a macromolecule with a unique sequence and function.The DNA double helical structure as deduced by,

1953 - James D. Watson and Francis Crickfrom the X-ray diffraction pattern of DNA, demonstrated by Rosalind Franklin and Maurice Wilkins, supported the template speculation.

The fundamental features of the DNA replication process have proved to be essentially identical in all species of the prokaryotic as well as the eukaryotic

worlds. Many of the protein complexes are functionally and structurally conserved.

3

DNA Stores Genetic Information Biochemical investigation of DNA1868 - Friedrich Miescher isolated a phosphorus-containing substance which he called “nuclein” from the nuclei of pus cells (leukocytes) obtained from discarded surgical bandages. He found nuclein to consist of an acidic portion (DNA) and a basic portion (protein). A similar acidic substance was also isolated from the heads of sperm cells. Meischer and others suspected that nuclein was associated with cell inheritance.

1944 - Oswald T. Avery, ColinMacLeod, and Maclyn McCarty produced the first evidence that DNA is the bearer of genetic information. They found that DNA extracted from a virulent strain of bacterium Streptococcuspneumoniae genetically transformed a non-virulent strain of this organism into a virulent form.

Injection of the encapsulated, virulent pneumococcus strain into mice showed lethality

while injection of the non-encapsulated, avirulentstrain had no effect.

Injection of heat-killed virulent streptococcus had no lethal effect.

Frederick Griffith had shown earlier that adding heat-killed virulent bacteria to a live

non-virulent strain permanently transformed the latter into lethal, virulent,

encapsulated bacteria

4

Avery, et. al. extracted the DNA from heat-killed virulent

pneumococci, removed the protein as completely as

possible, and added this DNA to non-virulent pneumococci. The DNA gained entrance into the

non-virulent bacteria, which were permanently transformed into a

virulent strain.

Avery and his colleagues concluded that the DNA extracted from the virulent strain carried the inheritable genetic message for virulence.

Others were skeptical about this conclusion and thought the protein contaminant in the DNA preparation may be the genetic material carrier. This possibility was ruled out by the following observations:

Treatment of the DNA preparation with proteases did not destroy itstransforming activity;

Treatment of the same with deoxyribonucleases did destroy the transforming activity of the DNA preparation.

1952 – Alfred D. Hershey and Martha Chaseperformed an experiment that provided independent evidence that DNA carries genetic information.Experiment:Bacteriophage (virus) cultures, in which either the

protein capsule is labeled with radioactive sulfur or the DNA core is labeled with radioactive phosphorus, are allowed to infect bacteria.

Agitation in a blender dislodges phage particles from bacterial cells.

Centrifugation concentrates cells, separating them from the phage particles left in the supernatant.

Results:Radioactive sulfur is found predominantly in the

supernatant. Radioactive phosphorus is found predominantly

in the cell fraction, from which a new generation of infective phage can be isolated.

Conclusion: The active component of the bacteriophage that transmits the infective characteristic is the DNA. There is a clear correlation between DNA and genetic information.

5

Replication of DNA as suggested by Watson and Crick

From their model, Watson and Crick could predict, well in advance of the actual experimental evidences, that the DNA double helical structure could be replicated by:1) separating the two strands, and2) synthesizing a complementary strand for

each. The separated parent strands would serve as the templates.

DNA Replication Follows a Set of Fundamental Rules:

DNA Replication is Semiconservative

Replication Begins at the Origin and Usually Proceeds Bidirectionally

DNA Synthesis Proceeds in a 5’→3’ Direction and is Semidiscontinuous

6

THREE HYPOTHESES FOR DNA REPLICATION

WEAVER

SEPARATION OF DNAS BY CESIUM CHLORIDE DENSITY GRADIENT CENTRIFUGATION

(a) Photo of DNA in an ultracentrifuge tubemade with UV light

(b) Densitometric trace of UV scan

7

PREDICTED DENSITIES OFNEWLY REPLICATED DNAMOLECULES ACCORDING

TO THE THREE HYPOTHESESABOUT DNA REPLICATION

WEAVER

DNA Replication is Semiconservative

Messelson-Stahl Experiment:Cells were grown for many generation in

a medium containing only 15N (15NH4Cl) as the nitrogen source, so that all the DNA was 15N labeled and showed up as a single band (blue) when centrifuged in a CsCl density gradient.

The cells were next transferred to a medium containing only 14N and allowed to grow for one generation. The DNA isolated from these cells banded in an intermediate region (between 15N DNA and 14N DNA).

Continuation of replication for a second generation yielded two hybrid DNA and two light DNAs (red), confirming semi-conservative replication.

Each DNA strand serves as a template for the synthesis of a new strand, producing two new DNA molecules, each with one new strand and one old strand. This is semi-

conservative replication.

8

Following the confirmation of a semiconservative mechanism of replication, a host of questions arose:

Are the parent DNA strands completely unwound before each is replicated?

Does replication begin at random places or at a unique point?

After initiation at any point in the DNA, does replication proceed in one direction or both?

9

Replication Begins at the Origin and Usually Proceeds Bidirectionally

E.coli cells were grown in a medium containing 3H-thymidine.

The cells were then lysed very gently to avoid fragmentation of DNA and the latter was allowed to settle onto a support that was overlaid with photographic emulsion for several weeks.

The radioactive thymidine residues generated tracks of silver grains in the emulsion, producing an image of the DNA molecule (an autoradiogram).

Non-replicating chromosome of E. coliwas a single huge circle of 1.7 mm long.

Visualization of bidirectional DNA replicationJohn Cairns Experiment

Replicating E. coli chromosome produced a structure resembling the Greek letter θ.

Cairns concluded that the loop resulted from the formation of two radioactive daughter strands, each complimentary to a parent strand.

Is one or are both ends of the loop dynamic points, termed replication forks, where parent DNA is being unwound and the separated strands quickly replicated?

Cairns results demonstrated that both DNA strands are replicated simultaneously.

A little variation on Cairns experiment indicated that replication of bacterial chromosomes is bidirectional: both ends of the loop have active replication forks.

Addition of high level of radioactivity (3H) for a short period just before the reaction was stopped allowed a distinction to be made between unidirectional and bidirectional replications, by determining whether label (red) was found at one or both replication forks in the autoradiogram. This technique has proved bi-directional replication.

Replication Begins at the Origin and Usually Proceeds Bidirectionally(contd.)

10

DETECTION OF BIDIRECTIONAL DNA REPLICATIONDNA synthesis in Bacillus subtilis spores was initiated synchronously and labeledfor a short time with a low level of radioactivity and then pulsed with a high level

of radioactivity; an interpretation of the autoradiograph is shown at right

WEAVER

Replication Begins at the Origin and Usually Proceeds Bidirectionally(contd.)

Partially Denatured DNAIf denaturation conditions are carefully controlled, regions that are rich in A=T base pairs will specifically denature while most of the DNA remains double-stranded. Such denatured regions, called bubbles, can be visualized with electron microscopy

Does the replication loop originate at a unique point in the DNA?Ross Inman and colleagues developed a technique called denaturation

mapping to answer this question. Inman showed that 48,502 bp chromosome of the bacteriophage λ could be

selectively denatured at sequences usually rich in A=T base pairs, generating a reproducible pattern of single-strand bubbles.

Isolated DNA containing replication loops can be partially denatured in the same way.

Using the denatured regions (bubbles) as points of reference this technique allows the position and progress of the replication forks to be mapped and measured.

The technique revealed that the replication loop always initiated at a unique point which was termed an origin.

11

THREE POSSIBLE WAYS IN WHICH DNA MIGHT BE SYNTHESIZEDAT THE REPLICATION FORK

TOP STRAND GROWS 3’ TO 5’BOTTOM STRAND GROWS 5’ TO 3’

BOTH STRANDS GROW 5’ TO 3’;LEADING STRAND (BLUE) GROWSCONTINUOUSLYLAGGING STRAND (RED) GROWSDISCONTINUOUSLY

BOTH STRANDS GROW 5’ TO 3’;BOTH STRANDS GROWDISCONTINUOUSLY

WEAVER

DNA Synthesis Proceeds in a 5’→3’ Direction and is Semidiscontinuous

Okazaki’s model of semidiscontinuous replication made two predictions:

(1) Since at least half of the newly synthesized DNA appears first as short pieces, one ought to be able to label and catch these before they are stitched together by allowing only very short periods (pulses) of labeling with a radioactive DNA precursor.

(2) If one eliminates the enzyme responsible for stitching together the short pieces of DNA (DNA ligase), these short pieces ought to be detectable even with relatively long pulses of DNA precursor.

Okazaki’s team tested experimentally his predictions by choosing replication of phage T4 DNA as it is simple and T4 DNA ligase mutant was available.

12

WEAVER

DNA Synthesis Proceeds in a 5’→3’ Direction and is Semidiscontinuous (contd.)

Replicating phage T4 DNA was labelled with very short pulses of radioactive DNA precursor.

The product DNAs were separated according to size by ultracentrifugation.

At the shortest times, the label went primarily into short DNA pieces (found near the top of the tube), as the discontinuous modelpredicted.

When mutant phage, with a defective DNA ligase gene, was used short DNA pieces accumulated even after relatively long labelling times.

A new DNA strand (red) is always synthesized in the 5’→3’ direction.The template is read in the opposite direction, 3’→5’. The leading strand is

continuously synthesized in the direction taken by the replication fork.The other strand, the lagging strand is synthesized discontinuously in short

pieces (Okazaki fragments) in a direction opposite to that in which the replication fork moves.

The Okazaki fragments are spliced together by DNA ligase.In bacteria, Okazaki fragments are ~1000 to 2000 nucleotides long. In eukaryotic

cells, they are 150 to 200 nucleotides long

DNA Synthesis Proceeds in a 5’→3’ Direction and is Semidiscontinuous (cond.)

13

DNA is Synthesized by DNA Polymerases1955 – the search for an enzyme that could synthesize DNA began.Arthur Kornberg and his colleagues initially purified and

characterized a DNA polymerase from E. coli.This polymerase, now called, DNA Polymerase I, is a single

polypeptide enzyme – Mr 103,000 – encoded by the polAgene.

Later investigators found that E. coli contain more distinct polymerases.

Search for other DNA polymerases began soon after the discovery of DNA pol I since evidence began to accumulate that it is not suitable for replication of the large E. coli chromosome on the following grounds:

the rate at which it adds nucleotides is too slow to account for the rates at which the replication fork moves in the bacterial cell;

DNA polymerase I has a relatively low processivity;

genetic studies demonstrated that many genes and therefore manyproteins, are involved in replication. Hence, DNA pol I does not act alone;

in 1969 John Cairns isolated a bacterial strain with a mutation in the DNA pol I gene that produced an inactive enzyme. This strain was however, viable.

A search for other DNA polymerases led to the discovery of E. coli DNA polymerase II and III in the early 1970s and DNA polymerase IV and V in 1999.

DNA polymerase III is the principal replication enzyme in E. coli.

14

Elongation of a DNA Chain(dNMP)n + dNTP → (dNMP)n+1 + PPi

DNA LengthenedDNA

The fundamental reaction is a phosphoryl group transfer.The nucleophile is the 3’-hydroxyl group of the nucleotide at the 3’-end of the

growing strand.Nucleophilic attack occurs at the α phosphorus of the incoming deoxy-

nucleoside 5’-triphosphate.The reaction appears to proceed with only a minimal change in free energy -

one phosphodiester bond is formed at the expense of a somewhat less stable phosphoanhydrie bond. Further stabilization of the lengthened DNA product is

achieved by the following factors:base-stacking interactions;base-pairing interactions;subsequent hydrolysis of the

PPi product by the enzyme pyrophosphatase.

• DNA synthesis is an example of tail-growth polymerization in which the nucleophile is at the growth end of the polymer and the attack is made on an activated form of the new monomer to be incorporated. Another example of this type of polymerization is in glycogen synthesis.

• In head-growth polymerization, it is the monomer that provides the nucleophile that attacks an activated site on the growing polymer. This occurs in both protein and fatty acid synthesis.

Basic features of polymerization actions

15

DNA polymerases are remarkably similar in overall shape, although they differ substantially in detail.

The polymerase unit resembles a right hand with fingers (blue), palm (yellow), and thumb (red).

The finger and the thumb domains wrap around DNA and hold it across the enzyme’s active site, which comprises residues primarily from the palm domain.

The first DNA polymerase structure determined was that of a fragment of E. coli DNA polymerase I called the Klenowfragment.

DNA Polymerase Structure

Requirement of Two Mg2+ in Polymerase Reaction

All the DNA polymerases catalyze the same polymerase reaction, which is dependent on two metal ions.

One metal ion binds both dNTP and 3’-OH of the primer, whereas the other interacts only with dNTP.

The two metal ions are bridged by the carboxylate groups of two aspartate residues in the palm domain of the polymerase. The side chains hold the metal ions in proper position and orientation.

The metal ion bound to the 3’-OH of the primer, activates it and facilitates its attack on the α-phosphate group of the dNTP substrate at the active-site.

The two metal ions together help stabilize the negative charge that accumulates on the pentacoordinate transition state..

The metal ion initially bound to the dNTP stabilizes the negative charge on the pyrophosphate.

16

DNA polymerase I actively requires a single unpaired strand to act as template, and a primer strand to provide a free –OH group at the 3’-end, to which a new nucleotide is added.

Most primers are oligonucleotides of RNA rather than DNA, and specialized enzymes synthesize primers when and where they are required.

Each incoming nucleotide is selected in part by base pairing to the appropriate nucleotide in the template strand.

The reaction product has a new 3’-OH group, allowing the addition of another nucleotide.

Requirements of DNA pol IPrimer

Primer terminus

Catalytic Mechanism of DNA pol I

Two Mg2+ ions are coordinately bonded to two phosphate groups of the incoming deoxynucleoside triphosphate and to three Asp residues, two of which are highly conserved in all DNA polymerases.

On of the Mg2+ ions (the top one in the figure) facilitates attack of the 3’-OH group of the primer on the αphosphate of the deoxynucleotide (dNTP).

The other Mg2+ ion (lower) facilitates the displacement of the PPi.

17

Replication is Very Accurate

Replication proceeds with an extraordinary degree of fidelity.

In E. coli a mistake is made only once for every 109 to 1010 nucleotides added. For the E. coli chromosome of ~4.6x106 bp, this means that an error occurs once per 1000 or 10,000 replications.

During polymerization, discrimination between correct and incorrect nucleotides relies not just on the hydrogen bonds that specify the correct pairing between complementary bases but also on the common geometry of the standard A=T and GΞC base pairs.The active site of DNA polymerase I accommodates only base-pairs with this geometry.

Contribution of base-pair geometry to the fidelity of DNA replication

An incorrect nucleotide may be able to hydrogen-bond with a base in the template, but it will generally not fit into the active-site.

Incorrect bases can be rejected before the phosphodiester bond is formed.

AccommodatedNot accommodatedAt the active site of DNA polymerase

Replication is Very Accurate (contd.)

A base can sometimes exist in the unusual tautomeric form, allowing it to hydrogen-bond with an incorrect partner.

Consequently the accuracy of the polymerization reaction itself will be insufficient to account for the high degree of fidelity in replication.

In fact, careful measurements in vitro have shown that DNA polymerase insert one incorrect nucleotide for every 104 to 105 correct ones.

A mechanism, intrinsic to virtually all DNA polymerases is a separate 3’→ 5’exonuclease activity that double-checks each nucleotide after it is added.

This nuclease activity of the polymerase, also called the proofreading activity, permits the enzyme to remove a newly added nucleotide and is highly specific for mismatched base-pairs.

Proofreading is not the opposite of polymerization reaction since pyrophosphate is not involved.

In the monomeric DNA pol I the polymerizing and proofreading activities have separate active sites within the same polypeptide.

When both selection and proofreading are combined, DNA polymerase leaves behind one error for every 106 to 108 bases added.

The measured accuracy of replication in E. coli is 109 to 1010. This additionalaccuracy is provided by a separate enzyme system that repairs mismatched base pairs remaining after replication.

18

Tautomers of

An example of error correction by the 3’→5’ exonuclease activity of

DNA polymerase I

DNA polymerase I is not the primary enzyme of replication.Instead it performs a host of clean-up functions during replication, recombination

and repair.DNA pol I has a special activity – 5’→3’ exonuclease activity (different from the

3’→5’ proofreading exonuclease activity) that is located in a structural domain that can be separated from the enzyme by mild protease treatment.

DNA pol I minus the 5’→3’ exonuclease domain is called the large fragment or the Klenow fragment (Mr 68,000).

Klenow’s fragment retains the polymerization and proofreading activities.The 5’→3’ exonuclease activity of DNA pol I can replace a segment of DNA (or

RNA) paired to the template strand in a process called nick translation. Indeed the removal of RNA primers from the 5’-ends of the newly synthesized DNA strands is achieved by the 5’→3’ exonucleaseactivity of RNA pol I. Other DNA pols lack this activity.

The Klenow fragment of DNA polymerase I

from the thermophilic bacterium Bacillus stearothermophilus. The active site for addition of nucleotides is deep in the crevice at the far end of the bound DNA. The dark blue strand is the template.

19

Nick Translation

In this process, an RNA or DNA strand paired to a DNA template is simultaneously degraded by the 5’→3’exonuclease activity of DNA polymerase I and replaced by the 5’→3’ polymerase activity of the same enzyme.

Nick translation by DNA pol I has a role in both DNA repair and removal of RNA primers during DNA replication.

The nick that remains after the DNA pol I dissociates is repaired by another enzyme called ligase.

DNA polymerase IIIA very complex DNA polymerase III, has ten types of subunits.The polymerization and proofreading activities reside in its α and ε subunits

respectively.The θ subunits associates with α and ε to form a core polymerase which can

polymerase DNA but with limited processivity.[After adding a nucleotide to a growing DNA strand, a DNA polymerase either dissociates or moves along the template and adds another nucleotide. Dissociation and Reassociation of the polymerase can limit the overall

polymerase rate – the process is much faster when a polymerase adds more nucleotides without dissociating from the template. The average number of nucleotides added before a polymerase dissociates defines its processivity.]

20

Two core polymerases can be linked by another set of subunits, a clamp-loading complex, or γ-complex, consisting of five subunits of four different types, τ2γδδ’.

The core polymerases are linked together by the τ subunits.

Two additional subunits χ and ψ are bound to the clamp loading complex or the γ-complex.

The γ subunit is generated by a translational frameshiftingmechanism of the τ gene that leads to premature translational termination.

DNA polymerase III

The entire assembly of 13 protein subunits [(αεθ)2τ2γδδ’χψ] is called DNApolymerase III*.

DNA polymerase III* can polymerize DNA but with a much lower processivity.The necessary increase in processivity is provided by the addition of the β

subunits, four of which complete the DNA polymerase holoenzyme.The β subunits associate in pairs to form donut-shaped structures that encircle the

DNA and act like clamps. One dimeric clamp per core subassembly.

Side View End View

Two β subunits of E. coli polymerase III (one red and one yellow) form a circular clamp that surrounds the DNA.

The clamp slides along the DNA molecule, increasing the processivity of the polymerase III holoenzyme to greater than 500,000 by preventing its dissociation from the DNA.

In the side view, surface contour models of the β subunits surround a space filling model of a DNA double helix.

The end-on view shows ribbon diagram of the two β subunits along the periphery and the DNA double helix space-filling model at the center.

21

DNA Replication Requires Many Enzymes and Protein FactorsThe replication process in E. coli requires along with the DNA polymerase III many

other different enzymes and proteins, each performing a specific task. The entire complex is termed the DNA replicase system or replisome.

Helicases are enzymes that move along the DNA and separate the strands that are to act as templates for the synthesis of the leading and the lagging strands. ATP hydrolysis is required to accomplish this task.

Topoisomerases relieves the topological stress in the helical DNA structure created by the strand separation.

The separated strands are stabilized by the DNA-binding proteins.Primases synthesize the primers, generally short segments of RNA, an absolute

requirement of the DNA polymerases.DNA polymerase III carries out the polymerase activity which includes three

different stages – initiation, elongation and termination.RNA primers are finally removed by the DNA polymerase I activity (5’→3’

exonuclease activity) and also fills up the gap created by the primer removal by its polymerase action.

The nick created by the DNA polymerase I action (between the newly synthesized Okazaki fragments) are sealed by the DNA ligase.

All these processes require coordination and regulation, an interplay best characterized in the E. coli system.

Why primers are made of RNA and not DNA?

DNA polymerases need a primer (more appropriately a 3’-OH group to start a nucleophillic attack on the α-phosphate group of the incoming nucleotide).

A primer synthesizing enzyme could have made either a DNA primer or a RNA primer.

The reason only RNA primers are made by a special RNA polymerase, primase(DNA-directed RNA polymerase) is that the primers are made with more errors since their synthesis is not subject to proof reading.

Making primers out of RNA guarantees that they will be recognized, removed and replaced with DNA by extending the neighboring Okazaki fragment.

The latter process is relatively error-free, because it is catalyzed by DNA pol I, which has a proofreading function.

22

Replication of the E. coli Chromosome Proceeds in Stages:Initiation

Elongation Termination

Replication process in E. coli was deciphered by reconstituting in vitro, the complex, multi-enzyme systems that direct chromosomal replication in cell free systems, purifying the component enzymes and carrying out mechanistic experiments.

The principles are highly conserved in all replication systems.

Arrangements of sequences in the E. coli replication origin , oriC

At oriC there are two series of short repeats – three repeats of a 13 bp sequence and four repeats of 9 bp sequence.

The repeated sequences are not identical. Certain nucleotides are particularly common in each position, forming a consensus

sequence.In positions where there is no consensus, N represents any of the four nucleotides.The arrows indicate the orientation of the nucleotide sequences.

Model for initiation of replication at the E. coliorigin, oriC

About 20 DnaA protein molecules, each with a bound ATP, bind at the four 9bp repeats in the oriC. The DNA is wrapped around the complex.

The three A=T-rich 13 bp repeats are then denatured sequentially. This require hydrolysis of ATP as well as participation of the bacterial histone-like protein HU.

The DnaC protein then loads the DnaB onto the unwound region.

Two ring-shaped hexamers of DnaB, one loaded onto each DNA strand, act as helicases, unwinding the DNA bidirectionally and creating two potential replication forks.

Many molecules of single-stranded DNA-binding protein (SSB) bind cooperatively to single-stranded DNA, stabilizing the separated strands and preventing renaturation.

DNA gyrase or DNA topoisomerase II relieves the topological stress incurred on the DNA structure due to unwinding by DnaB helicase.

Initiation is the only phase of DNA replication that is regulated such that replication occurs only once in each cell cycle. Mechanism is not well understood.

23

RNA polymerase requirement:RNA could be required to read into the origins from adjacent transcription units; by

terminating at sites in the origin, it could provide the 3’-OH ends that prime DNA polymerase III. (e.g. D-loops at the mitochondrial origin)

Alternatively, the act of transcription could be associated with a structural change that assists initiation. This idea is supported by observation that transcription does not have to proceed into the origin; it is effective up to 200 bp away from the origin, and can use either strand of DNA as template in vitro.

Probable mechanisms of regulationThe oriC DNA is methylated by the Dam (DNA adenine methylation) methylase, which

methylates at the N6-position of adenine within the palindromic sequence (5’)GATC. The oriC region is rich in the GATC sequence – 11 in 245 bp, normal occurance - 1 in 256 bp.

Immediately after replication the DNA is hemimethylated at oriC which is sequestered for a period by interaction with the plasma membrane.

When released from the PM oriC must be fully methylated before it can again bind DnaA.

Slow (timescale of 20 to 40 min.) ATP hydrolysis by DnaA protein, which cycles the protein between active (ATP-bound) and inactive (ADP-bound) forms also plays a role in regulation of initiation.

Does methylation at the origin regulate initiation?The ability of a plasmid relying upon oriC to replicate in dam- E. coli depends on its

state of methylation:If the plasmid is methylated it undergoes a single round of replication, and

then the hemimethylated product accumulates.Thus a hemimethylated origin cannot be used to initiate a replication

cycle.Why a hemimethylated origin fails to initiate replication?

Initiation may require full methylation of the Dam target sites in the origin.Initiation may be actually inhibited by hemimethylation of the oriC site.

The answer is the second case since, in vitro, an origin of non-methylated DNA can function effectively.

24

Probable mechanisms of regulation (contd.)

The GATC sites at the origin remain hemimethylated for ~13 minutes after replication. This long period is unusual because at typical GATC sites elsewhere in the genome replication begins immediately (<1.5 min.) following replication.

One other region behaves like oriC - the promoter of the dnaA gene also shows a delay before methylation begins. While it is hemimethylated, the dnaApromoter is repressed which causes a reduction in the level of DnaA protein – the critical initiator protein.

What is/are responsible for the delay in methylation at oriC and dnaApromoter?

The most likely explanation is that these regions are sequestered in a form in which they are inaccessible to the Dam methylase.

SeqA, the product of the gene seqA whose mutation leads to the reduction in the delay of methylation at both oriC and dnaA promoter, binds strongly to hemimethylated DNA. Continued presence of SeqA prevents formation of an open complex at the origin.

The presence of SeqA is also an absolute requirement for the association of the hemimethylated oriC with membrane.

Probable mechanisms of regulation (contd.)

Membrane association of the hemimethylated oriC prevent re-initiation:- indirectly by sequestering the origin from the action of Dam methylase;- directly by some component at the membrane that inhibits the reaction. There is evidence for such a component in the membrane fraction and it competes with DnaA.

In in vitro experiment DnaA association with phospholipids (probably with PM too) promote the exchange of ATP with ADP bound to DnaA.

Also availability of DnaA for binding to the origin regulates DNA replication initiation.

The full scope of the system used to control re-initiation is not clear, but several mechanisms may be involved:● physical sequestration of the origin;● delay in re-methylation;● inhibition of DnaA binding;● repression of dnaA transcription.

25

Watson

Watson

26

The elongation phase of replicationincludes synthesis of the leading and the lagging strands.

Leading strand synthesis is straight forward. Primases (DnaGprotein) synthesizes a short (10 to 60 nucleotides) RNA primer at the replication origin.

Deoxyribonucleotides are added to primer by DNA polymerase III.

Leading strand synthesis proceeds continuously, keeping pace with the unwinding of DNA at the replication fork.

Lagging strand synthesis is accomplished in short Okazaki fragments.Lagging strand synthesis formally proceeds in the opposite direction from fork

movement.At intervals, primase synthesizes an RNA primer for a new Okazaki fragment.Each primer is extended by DNA polymerase III.DNA synthesis continues until the fragment extends as far as the primer of the

previously added Okazaki fragment.A new primer is synthesized near the replication fork to begin the process again.Okazaki fragment synthesis seems straightforward, but in reality, is quite complex.

REPLICATION AT PERMISSIVE TEMPERATURE: BLUEREPLICATION AT NON-PERMISSIVE TEMPERATURE: RED

WEAVER

A loss of function mutation in the helicase gene that participate in DNA replication would be lethal.

One way to generate mutants with defects in essential gene is to make the mutation conditional, usually temperature sensitive (ts).

In 1968, Francois Jacob et. al. discovered two classes of tsmutants in E. coli DNA replication.

EFFECTS ON DNA REPLICATION OF TWO TEMPERATURE-SENSITIVE MUTANTSOF E. coli DNA HELICASE

One of the type I mutants that showed an immediate shut-off of DNA synthesis on raising the temperature from 300C to 400C was the dnaB mutant.

The dnaB product was known to be an ATPase and was found to be associated with the primase, which makes primers for DNA replication.

These findings suggested that DnaB is the DNA helicase that unwinds the DNA double helix during E.coli DNA replication.

27

DNA HELICASE ASSAY

WEAVER

To demonstrate that DnaB has DNA helicase activity

Linear

Circular

LeBowitz and McMacken made a helicase substrate by 32P-labelling a single-stranded 1.06 kb DNA fragment at its 5’-end (red) and annealing the fragment to an unlabeled single-stranded recombinant M13 DNA (blue) bearing a complemetary 1.06 kb region.

The DnaB protein or any DNA helicase, can unwind the double-stranded region of substrate and liberate the short piece of DNA (red) from its longer, circular partner.

WEAVER

DNA HELICASE ASSAY

ALTHOUGH PRIMASE(DnaG) AND SINGLE-

STRAND BINDING PROTEIN(SSB) BOTH STIMULATEDNA HELICASE (DnaB),

NEITHER HAVE HELICASEACTIVITY OF THEIR OWN

DnaB is the helicase that unwinds the DNA at the replication fork

28

PRIMING OF DNA SYNTHESIS BY SHORT SEQUENCES OF RNA (RED)

DNA POLYMERASE USES THE PRIMERS AS STARTING POINTS TO SYNTHESIZE PROGENY DNA STRANDS (GREEN ARROWS)

WEAVER

The first line of evidence supporting RNA priming was the finding that replication of M13 phage DNA by an E. coli extract is inhibited by the antibiotic rifampicin, which inhibits E. coli RNA polymerase and not DNA polymerase.

The evidence towards RNA priming as a more generalized phenomenon came from a different experiment carried out by Tuneko Okazaki (next slide).

DNase cannot completely destroy Okazaki fragments was perhaps the best evidence.

WEAVER

Lanes a-d: before DNaseLanes e-h: after DNaseLanes a,e: defective in RNase HLanes b,f: defective in DNA Pol ILanes c,g: defective in bothLanes d,h: wild-type

DETECTION OF RNA PRIMERS ●Tuneko Okazaki and colleagues isolated Okazaki fragments from wild-type and mutant E. coli cells lacking one or both of the nucleases (RNase H and DNA pol I) that degrade RNA primers.

●Next they labeled the intact primers on the Okazaki fragments with [32P]GTP and a capping enzyme. Only intact primers will show up.

●They destroyed the DNA in the fragment with DNase, leaving only the labeled primers.

●They subjected these primers to electrophoresis and detected their positions by autoradiography.

●The best yield of primers occurred when both nucleases were defective.

●The primers in all cases were 11+1 ntlong.

29

WEAVER

A GENERAL SCHEME OF THE ROLLING-CIRCLE MODEL OF DNA REPLICATIONLEADING TO THE PRODUCTION OF SINGLE-STRANDED PROGENY MOLECULES

WEAVER: FIG. 21.1

Speed of Replication

Mock and Marians started with the 6702-nt plus (+) strand (red) from the f1 phage and annealed it to a primer (green) that hybridized over a 282-nt region (yellow).

The primer contained a primosome binding site (orange).

The primer was elongated in presence of 32P-dNTPs with DNA pol III holoenzymeand SSB to create the negative strand (blue).

The product was a ds-template for multiple rounds of rolling circle replication, in which the free 3’-end could serve as the primer.

30

WEAVER: FIG. 21.2

(The template contained a 32P-labelled, tailed, full-length strand with a free 3’-OH group for priming.)•MEASURMENT OF RATE OF DNA SYNTHESIS IN VITRO

(a) POL III + ‘PREPRIMOSOMAL PROTEINS’ (primosomal – DnaG) + SSB(b) POL III + DNA HELICASE

•GELS SHOW CHAIN EXTENSION IN KB AT 10-SEC INTERVALS•BOTH ANALYSES GAVE INCORPORATION RATE OF ~730 NTS/SEC (RIGHT)

Measurement of the Rate of Fork Movement in vitro

The complexity in Okazaki fragment synthesis lies in the coordination of leading and lagging strand synthesis: both strands are produced by a single asymmetric DNA polymerase III dimer, which is accomplished by looping the

DNA of the lagging strand, bringing together the two points of polymerization.

Black arrows: direction of movement of the parent DNA through the complex.Red arrows: direction of the DNA synthesis.

31

The DnaB helicase and DnaG primase constitutes a functional unit within the replication complex, the primosome.

The DnaB helicase unwinds the DNA at the replication fork as it travels along thelagging strand in the 5’→3’ direction.

DNA primase occasionally associates with DnaB helicase and and synthesize a short RNA primer.

A new β sliding clamp is then positioned at the primer by the clamp-loading complex of DNA polymerase III.

32

When synthesis of an Okazaki fragment has been completed, replication halts, and the core subunits of DNA polymerase III dissociate from their β sliding clamp and hence, from the completed Okazaki fragment.

The core subunit then associates with the new clamp.This initiates synthesis of a new Okazaki fragment.

33

Once an Okazaki fragment has been completed, its RNA primer is removed and replaced by DNA polymerase I.

The nick is sealed by DNA ligase.The entire complex responsible for coordinated DNA synthesis at a replication fork

is a replisome.

RNA primers in the lagging strand are removed by the 5’→3’exonuclease activity of DNA polymerase I and replaced with DNA by the same enzyme.

The remaining nick is sealed by DNA ligase.

DNA ligase catalyzes the formation of a phosphodiesterbond between a 3’ hydroxyl at the end of one strand and a 5’phosphate at the end of the other strand.

The phosphate must be activated by adenylation.

DNA ligase from bacteria uses NAD+ as a source of the activating AMP group whereas eukaryotes and viruses use ATP for the same.

DNA ligase has become an important reagent in recombinant DNA technology.

34

Mechanism of the DNA Ligase Reaction(from a Lys residue)

Eventually, the two replication forks of the circular chromosome meet at a terminus region containing multiple copies of a 20 bpsequence called Ter.

The Ter sequences are arranged on the chromosome to create a sort of trap that a replication fork can enter but cannot leave.

Termination

Tus (termination utilization sequence) binds at the Ter sequence.Only one Tus-Ter complex functions per replication cycle and hence, can

arrest replication fork from only one direction - decided by which of the two replication forks first encounter the complex.

When either replication fork encounters a functional Tus-Ter complex it halts. The other fork halts when it meets the first arrested fork.

The final few hundred base pairs of DNA between these large protein complexes are then replicated (mechanism not known).

Replication is completed giving rise to two topologically interlinked or catenatedcircular cromosomes known as catenanes.

35

E. coli type II topoisomerase(inflicts double-stranded breaks)

The separated chromosomes then segregate into daughter cells at cell division.

Bacterial Replication is originated in Membrane-bound Replication FactoriesOnce bidirectional replication is initiated at the origin, the two replisomes do not travel away from each other along the DNA. Instead, the replisomes are linked together and tethered to one point (center of the elongated cell) on the bacterial inner membrane,

and the DNA substrate is fed through the “replication factory.”

36

Acyclovir

Most DNA viruses encode their own DNA polymerases, and some of these have become targets for pharmaceuticals.

The DNA polymerase of the herpes simplex virus is inhibited by acyclovir, a compound developed by Gertrude Elion.

Acyclovir consists of guanine attached to an incomplete ribose ring.

It is a substrate for the virally encoded thymidine kinase with which it binds 200-fold stronger than the cellular enzyme. This confers specificity for the viral-infected cells and not the uninfected cells.

Acyclo-GTP (produced by action of cellular kinase on the Acyclo-GMP) is both a substrate and inhibitor (chain terminator) of the herpes DNA polymerase mainly.

WEAVER

A GENERAL SCHEME OF THE ROLLING-CIRCLE MODEL OF DNA REPLICATIONLEADING TO THE PRODUCTION OF SINGLE-STRANDED PROGENY MOLECULES

37

Bacteriophage M13

M13 belongs to the Inoviridae family and possess a simple helical capsids.The phage is about 900 nm long & 9 nm diameter & the particles contain five proteins.The major coat protein is the product of phage gene 8 (gp8) & there are 2700-3000 copies of this protein per particle, together with approximately five copies each of four minor capsid proteins, gp3, gp6, gp7 & gp9, located at the ends of the filamentous particle.Because the phage circular, single-stranded, +DNA (6408 bases) is packaged inside the core of the helical particle, the length of the particle is dependent on the length of the genome.In all Inovirus preparations, ''polyphagepolyphage'' (containing more than one genome length of DNA), ''miniphageminiphage'' (deleted forms containing 0.2-0.5 phage genomes length of DNA), & ''maxiphagemaxiphage'' (genetically defective forms but containing more than one phage genome length of DNA) occur.The plastic property of these filamentous particles has been exploited by molecular biologists to develop the M13 genome as a cloning vector - insertion of foreign DNA into the genome results in recombinant phage particles which are longer than the wild-type filaments.Unlike most viruses, there is no sharp cut-off at which the genome can no longer be packaged into the particle.

Bacteriophage M13 (contd.)

38

Inovirus phages are 'male-specific', i.e. they require the F pilus on the surface of Escherichia coli for infection.On infection, an interaction between gp3 located at one end of the filament together with gp6, & the end of the F pilus.This interaction causes a conformational change in gp8.Initially, its structure changes from 100% α-helix to 85% α-helix, causing the filament to shorten.The end of the particle attached to the F pilus flares open, exposing the phage DNA.Subsequently, a second conformational change in the gp8 subunits reduces its α-helical content from 85% to 50%, causing the phage particle to form a hollow spheroid ~40 nm in diameter & expelling the phage DNA, initiating infection of the host cell.Following infection the circular, ss-(+)DNA is converted to the replicative form by the host machinery. The (–)strand of the RF serves as the template for replication.Replication takes place by a rolling-circle model but no concatemer is formed.Virus assembly occurs at the cell membrane. Fully formed virions bud out from the surface and are extruded out into the cell environment. M13 is thus an unique lyticphage that undergoes intracellular proliferation without causing lysis of the host cell.

Bacteriophage M13 (contd.)

M13 SS to RF synthesis: Paradigm for leading strand synthesis

Required factors:SSB, tetrameric single-strand binding protein: binds

ssDNA cooperativelyRNA polymerase, synthesizes RNA primer at

hairpinPol III holoenzyme, synthesizes complementary

strand, forming RFII (open circle)Pol I, remove RNA primer, extends 3’ end of

complementary strand to form a nickLigase/DNA gyrase, seals nick to yield RFI

(covalently supercoiled circle)

RF = replication form

57 nucleotude long palindromic sequence not covered by SSB

39

φx174First ever organism to be sequenced by Sanger, et. al. in 1978.A small bacteriophage (MW 2.7x105 D)Icosahedral with 12 prominent spikes.Single-stranded, closed, circular genome having 5386 bases.Nine overlapping genes (first suspected when the total protein coding ability of the

phage was found to exceed the expected normal genome size).Four of the nine genes code for structural proteins (gpF and gpJ for capsid; gpG

and gpH for spikes).Infection of the host takes place via the interaction between the spikes and

lipopolysaccharide receptors on the host cell surface.

A

A*C

D

EJ

F

G

H

B K

OR+

1/5386

1000

2000

3000

4000

5000

φx174

gpA and gpA* are involved in turning off host DNA replication.

gpC is required for viral strand synthesis.Uses the host DNA synthetic machinery.gpJ is required for condensation of the viral genome

into the prohead during viral assembly.The prohead is assembled by interactions between gp

– B, D, F, G and H undergoes structural rearrangements with the elimination of the scaffolding proteins (gp B and D).

Transcription and translation occurs during the entire infection period.

Host cell is lysed to liberate the progeny virions.

φX174 SS to RF synthesis: Role of the Primosome

The + strand is coated with SSB except for a 44-nt hairpin near position 2300.A 70-nt sequence containing this hairpin known as pas ( primosome assembly site), is then recognized and bound by the PriA, PriB and PriC proteins.DnaB6.DnaC6 complex next add to the DNA with the help of the DnaT protein in an ATP requiring process.DnaC proteins is then released yielding the primosome.The primosome is propelled in the 5’→3’ direction along the + strand by PriA- and DnaB-catalysed ATP hydrolysis. This motion which displaces the SSB in its path, is opposite to that of the template reading during DNA chain propagation

(-) strand replication: paradigm for lagging strand synthesis

40

φX174 SS to RF synthesis: Role of the Primosome (contd.)

+ DnaG

At randomly selected sites, the primosome reverses its migration while primase (DnaG) synthesizes an RNA primer.

The initiation of primer synthesis requires the participation of DnaB which, through concomitant ATP hydrolysis, is thought to alter template DNA conformation in a manner required by primase.

Pol III holoenzyme extends the primers to form Okazaki fragments.

Pol I excises the primers and replaces them by DNA ligase and

supercoiled by DNA gyrase to form the φx174 RF.The primosomeremains complexed

with the DNA where it parti-cipates in + strand synthesis

φX174 (+) strand replication serves as a model for leading strand synthesisLooped rolling circle mode

+ strand synthesis begins with the primosome-aided binding of the gpA protein to a 30-bp recognition site. gpA specifically cleaves the phosphodiesterbond preceding + strand nucleotide 4306.

Rep protein attaches to the – strand at the gpA and with aid of the primosome associated with the + strand commences unwinding the duplex DNA from the + stand’s 5’-end.

The displaced + strand is coated with the SSB.

Pol III holoenzyme extends + strand from the free3’-OH group.

The extension process generates a looped rolling circle structure.

When it has become a full circle, gpA protein again makes a specific cut at the replication origin so as to form the covalent linkage with the new + strand 5’-end

41

ROLLING-CIRCLE MODEL OF BACTERIOPHAGE λ DNA REPLICATIONTHIS SCHEME RELATES TO THE SYNTHESIS OF DOUBLE-STRANDED DNA

WEAVER

A rolling circle mode of replication is seen both during replication of bacteriophage M13 and φx174 where only a single ccc-ss copy is

produced each time and in the replication of bacteriophage lambda where ds-linear concatemer (multiple copies of a genome ) is produced.

λ (lambda) phageFamily – Siphoviridae.Host – K12 strain of Escherichia coli.Structure – icosahedral head of 55 nm in diameter and a non-contractile tail

with a thin tail fiber at its end. Genome – double-stranded, linear DNA molecule with cohesive ends of 12

nucleotides long, that have complementary base sequences and canpair with each other (cos sites). By virtue of their cohesive ends, following infection the linear genome circularizes. E. coli DNA ligaseseals the break. Over 40 genes have been mapped in the λ genome that are clustered according to their function.

PP

GGGCGGCGACCT

CCCGCCGCTGGA 5’5’ 3’

3’

Circularization & Ligation

T G

A C

42

Phage λ concatemers are generated by rolling-circle replication.

Rolling circle replication generates linear copies of a genome rather than circular copies.

A nick occurs in one of the two phosphodiesterbackbones.

A free 3' OH end is generated. This serves as a primer for DNA polymerase.

When the 3' OH end is extended (leading strand synthesis), the 5' end is displaced.

The displaced strand in turn, serves as a template for replication (lagging strand synthesis).

The consequence of this mechanism of replication is the production of concatemer copies of the circular molecule. Thus multiple copies of a genome are produced.

Phage λ concatemers are cut by nucleases that recognize specific sequences of the molecular ends, yielding non-permuted molecules with sticky or cohesive ends, also called cos-sites.

1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1

1’ 2’ 3’ 4’ 5’ 6’ 7’ 1’ 2’ 3’ 4’ 5’ 6’ 7’ 1’ 2’ 3’ 4’ 5’ 6’ 7’ 1’

4 5 6 7 1 2 3

2’ 3’ 4’ 5’ 6’ 7’ 1’

Production of mature viral DNA molecules from concatemers:

In λ-bacteriophage nucleases make staggered cuts on the two strands of a concatemer, recognizing specific sequences, thereby generating molecules with cohesive or sticky ends.

43

Identification of Two Origins of Replication in the Single Chromosome of theArchaeon Sulfolobus solfataricus

Nicholas P. Robinson1, Isabelle Dionne1, 3, Magnus Lundgren2, 3, Victoria L. Marsh1, Rolf Bernander2 and Stephen D. Bell , 1, ,

1 Medical Research Council Cancer Cell Unit, Hutchison MRC Research Centre, Hills Road, Cambridge CB2 2XZ, United Kingdom2 Department of Molecular Evolution, Evolutionary Biology Center, Uppsala University, Norbyvägen 18C, SE-752 36, Uppsala, Sweden

Cell (2004) 116 (1), p 25-38

AbstractEukaryotic chromosomes possess multiple origins of replication, whereas bacterial chromosomes are replicated from a single origin. The archaeon Pyrococcus abyssialso appears to have a single origin, suggesting a common rule for prokaryotes. However, in the current work, we describe the identification of two active origins of replication in the single chromosome of the hyperthermophilic archaeon Sulfolobussolfataricus. Further, we identify conserved sequence motifs within the origins that are recognized by a family of three Sulfolobus proteins that are homologous to the eukaryotic initiator proteins Orc1 and Cdc6. We demonstrate that the two origins are recognized by distinct subsets of these Orc1/Cdc6 homologs. These data, in conjunction with an analysis of the levels of the three Orc1/Cdc6 proteins in different growth phases and cell cycle stages, lead us to propose a model for the roles for these proteins in modulating origin activity.

Multiple origins of replication in archaea

Lori M. Kelmana and Zvi Kelmanb, aMontgomery College, 20200 Observation Drive, Germantown, MD 20876, USAbUniversity of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology, 9600 Gudelsky Drive, Rockville, MD 20850, USA

Trends Microbiol (2004) 12 (9), p 397-401

44

A comparison of chromosomal DNA replication in the three domains of lifelk and lk

PolBPolD and/or PolBgPolCPolymeraseRFCRFCγ-complexClamp loaderPCNAPCNAfβ subunitSliding clampPolα/PrimasePrimaseDnaGPrimase

RPASSB or RPAeSSBSingle-stranded DNA-binding protein

Cdc6 and Cdt1dCdc6/ORCcDnaA and DnaCHelicase loaderMCMMCMDnaBHelicaseORCCdc6/ORCcDnaAOrigin recognitionMultipleSingle or multipleSingleReplication origin(s)LinearCircularLinear or circularChromosomeEukaryaArchaeaBacteria

a Bacterial-like features and proteins are in red, eukaryal-like features and proteins are in blue and archaeal-specific proteins are in green.b Abbrevations: MCM, mini-chromosome maintenance; ORC, origin recognition complex; PCNA, proliferating cell nuclear antigen; RFC, replication factor C; RPA, replication protein A; SSB, single-stranded DNA-binding protein.c As eukaryotic Cdc6 is similar to subunits of ORC, and the function of the archaeal homologs are not yet clear, they are referred to as Cdc6/ORC homologs.d Other factors might also be involved.e Depends on the organism.f In the euryarchaeal species PCNA is a single polypeptide, whereas in the crenarchaeal species three polypeptides have been identified.g In the euryarchaeal species in which PolD was identified it is not yet clear whether it or PolB is the replicativeenzyme.

DNA replication: (prokaryotic – E. coli chromosome) DNA supercoiling – linking number, negative and positive supercoilng, topoisomerases, plectonemic and solenoidal supercoiling; DNA replication – semiconservative (Messelson-Stahl’s experiment), bidirectional (Cairns’ experiment), semidiscontinuous (Okazaki fragments); mechanism of replication – participating enzymes and proteins factors –dnaA and dnaC gene products, helicase, single-stranded binding proteins, topoisomerase, primase, DNA polymerase III, DNA polymerases I, ligase; rolling circle mode of replication; asymmetric replication – looped rolling circle - φX174 and M-13 bacteriophages; concatemer formation - λ bacteriophage.

☺☺☺HAPPY ENDING!☺☺☺