Chapter 11 DNA Replication

11.1, 11.2, 11.3, 11.4

DNA replication

the process in which original DNA strands are used as templates for the synthesis of new DNA strands.

During the replication process, the two complementary strands of DNA come apart and serve as [blank], or parental strands, for the synthesis of two new strands of DNA.

template strands

template strand

a strand of DNA that is used to synthesize a complementary strand of DNA or RNA.

the individual nucleotides have access to the template strands after…

…the double helix has separated. Hydrogen bonding between individual nucleotides and the template strands must obey the AT/GC rule.

to complete the replication process, a covalent bond is formed between a phosphate of one nucleotide and the sugar of the previous nucleotide. The two newly made strands are referred to as the [blank].

daughter strands

daughter strands

in DNA replication, the two newly made strands of DNA.

DNA is replicated in such a way that both copies…

retain the same information—the same base sequence—as in the original molecule.

In the late 1950s, three different mechanisms were produced for the replication of DNA:

conservative model, semiconservative model, and dispersive model.

conservative model

an incorrect model of DNA replication in which both parental strands of DNA remain together following DNA replication.

semiconservative model

the correct model for DNA replication in which the newly made double-stranded DNA contains one parental strand and one daughter strand.

dispersive model

an incorrect model for DNA replication in which segments of parental DNA and newly made DNA are interspersed in both strands following the replication process.

Matthew Meselson and Franklin Stahl

In 1958, they devised a method to investigate the different DNA models. They found a way to experimentally distinguish between daughter and parental strands.

Meselson and Stahl’s hypothesis:

Based on Watson’s and Crick’s ideas, DNA replication is semiconservative,

Meselson and Stahl’s experiment:

They grew Escherichia coli (E. coli) in a medium containing heavy nitrogen (N-15) for several generations. This caused the bacteria to incorporate the heavy nitrogen into their DNA, making it denser. After the bacteria had fully incorporated the heavy nitrogen, Meselson and Stahl transferred them to a medium containing light nitrogen (N-14). This allowed the bacteria to replicate their DNA with the lighter nitrogen. They then collected samples after one and two rounds of replication and used a cesium chloride density gradient centrifugation. This technique separated the DNA based on density.

Results after one generation in Meselson and Stahl’s experiment:

All of the DNA sedimented at a density was half-heavy. Both semiconservative model and dispersive models are consistent.

why did the results after the first generation was not consistent with the conservative model?

the conservative model predicts two separate DNA types: a light type and a heavy type. Because all of the DNA had sedimented as a single band, this model was disproved.

why did the results after the first generation was consistent with the semiconservative model?

the semiconservative model predicts the replicated DNA would contain one original strand (a heavy strand) and a newly made daughter strand ( a light strand).

why did the results after the first generation was consistent with the dispersive model?

in a dispersive model, all of the DNA should have been half-heavy after one generation as well.

Results after one generation in Meselson and Stahl’s experiment:

A mixture of light DNA and half-heavy DNA was observed. This result was consistent with the semiconservative model of DNA replication.

why did the results after the second generation was consistent with the semiconservative model?

some DNA molecules should contain all light DNA, and other molecules should be half-heavy, which was the result of the experiment.

why did the results after the second generation was not consistent with the dispersive model?

the dispersive model predicts that after two generations, the heavy nitrogen would be evenly dispersed among four strands, each strand containing ¼ heavy nitrogen and ¾ light nitrogen. However, this result was not obtained.

Result of Meselson and Stahl’s experiment:

The experiment provided compelling evidence in favor of only the semiconservative model for DNA replication.

the site on the bacterial chromosome where DNA synthesis begins is known as..

the origin of replication. Bacterial chromosomes have a single origin of replication.

the synthesis of new daughter strands is initiated within the origin and proceeds in both directions, or [blank], around the bacterial chromosome

bidirectionally

bidirectional

(1) the manner in which two replication forks move, in opposite directions outward from the origin; (2) refers to a regulatory element that can function in either the forward or the reverse direction.

replication fork

the region in which two DNA strands have separated and new strands are being synthesized.

two replication forks move in opposite directions outward from the origin. eventually, the replication forks meet each other on the opposite side of the bacterial chromosome to…

…complete the replication process.

oriC

origin of chromosomal replication for e.coli.

three types of DNA sequences are found within oriC:

an AT-rich region, DnaA box sequences, and GATC methylation sites.

AT-rich region

where DNA strand separation initially occurs.

DnaA boxes

binding sites for DnaA proteins needed to form replication forks.

GATC methylation sites

provide a mechanism for regulating DNA replication

the first step to form two DNA replication forks:

DnaA proteins bind to DnaA boxes, forming a complex with the origin
and with each other.

the second step to form two DNA replication forks:

Interaction with additional DNA-binding proteins causes the DNA to bend around the DnaA protein complex, which in turn produces mechanical stress that leads to strand separation in the A-T rich region.

the third step to form two DNA replication forks:

DnaA and DnaC proteins recruit DnaB protein (helicase enzyme) to the complex.

DnaB protein (helicase enzyme)

an enzyme that breaks the hydrogen bonds between double-stranded DNA.

the fourth step to form two DNA replication forks:

Bidirectional movement of helicase enzymes promotes strand separation and movement of the replication forks outward from oriC.

GATC methylation sites are involved in…

…regulating replication. It ensures only one round of replication.

DNA adenine methyltransferase (Dam)

methylates the adenine (A) on both strands. This full methylation facilitates the initiation of DNA replication at the origin.

Following DNA replication, the newly made strands are…

not methylated, because adenine rather than methyladenine is incorporated into the daughter strands.

The initiation of DNA replication at the origin does…

…not readily occur until after the GATC sites are fully methylated.

Because it takes several minutes for Dam to methylate the GATC sites within this region…

DNA replication does not occur again until after the methylation is completed.

Dna A proteins

Bind to DnaA box sequences within the origin of replication to initiate DNA replication.

DnaC proteins

Aid DnaA in the recruitment of DNA helicase to the origin.

DNA helicase (DnaB)

separates double-stranded DNA

DNA Gyrase (topoisomerase ll)

Removes positive supercoiling ahead of the replication fork.

single-strand binding proteins

Bind to single-stranded DNA and prevent it from reforming a double-stranded structure.

primase

synthesizes short RNA primers

DNA polymerase lll

synthesizes DNA in the leading and lagging strands

DNA polymerase l

Removes RNA primers, fills in gaps within DNA

DNA ligase

covalently attaches adjacent Okazaki fragments

Tus

binds to ter sequences and prevents the advancement of the replication fork.

unwinding the double helix

(1) DNA helicase separates the two DNA strands by breaking the hydrogen bonds
between them; (2) This generates positive supercoiling ahead of each replication fork; (3) Topoisomerase II also called DNA gyrase travels ahead of the helicase and
alleviates these supercoils; (4) Single-strand binding proteins bind to the separated DNA strands to keep them apart.

the next event in DNA replication is the synthesis of short strands of RNA (rather than DNA) called.

RNA primers.

RNA primer

a short strand of RNA, made by primase, that is used to elongate a strand of DNA during DNA replication.

These strands of RNA are synthesizes by the linkage of ribonucleotides via an enzyme known as

primase

in the leading strand, a single primer is…

…made at the origin of replication.

leading strand

a strand during DNA replication that is synthesized continuously toward the replication fork.

in the lagging strand…

…multiple primers are made.

lagging strand

a strand during DNA replication that is synthesized as short Okazaki fragments in the direction away from the replication fork.

there are 5 DNAP (DNA polymerase) enzymes in E.coli, we will discuss DNAP l and DNAP lll in this chapter. DNAP lll is used to…

…synthesize most of the DNA in the lading and lagging strands.

Structure of DNA lll:

(1) DAP III is a complex structure made up of 10 different subunits; (2) The complex containing all 10 subunits is called a holoenzyme; (3) The α subunit is the catalytic subunit where polymerase activity resides.

Structure of DNA l

(1) Composed of a single subunit; (2) Removes RNA primers and replaces them with deoxyribonucleotides.

Synthesis of Leading strand:

One RNA primer is made at the origin, and then DNA polymerase lll attaches nucleotides in a 5’ to 3’ direction as it slides toward the opening of the replication fork. The synthesis is continous.

Synthesis of the Lagging Strand:

(1) Synthesis is also in the 5’ to 3’ direction, however it occurs away from the replication fork; (2) Many RNA primers are required; (3) DNA pol III uses the RNA primers to synthesize small DNA fragments (1000 to 2000 nucleotides in bacteria, 100-200 in eukaryotes). These are termed Okazaki fragments after their discoverers; (4) Synthesis of the lagging strand is discontinuous.

Synthesis of the Lagging Strand cont:

(1) Removal of RNA primers; (2) Fill in the gap/replace with deoxyribonucleotides. Steps 1 and 2 performed by DNAP I. Uses a 5’ to 3’ exonuclease activity to digest the RNA and 5’ to 3’ polymerase activity to replace it with DNA (3) Covalently link adjacent Okazaki fragments. Step 3 requires by DNA ligase which catalyzes the formation of a
phosphodiester bond between Okazaki fragments.

DNA replication is…

bidirectional

At each replication fork, there is a [blank] and a [blank]

leading and lagging strand

Primosome

a protein complex that includes DNA helicase and primase. This complex leads the way at the replication fork.

Role of Primosome

separates the parental strands and synthesizing RNA primers a regular intervals along the lagging strand.

Replisome

a complex that consists of a primosome and dimeric DNA polymerase.

Two DNA pol lll proteins act in concert to replicate both the leading and lagging strands. The two proteins form a [blank] that moves as a unit toward the replication fork.

dimeric DNA polymerase

Dimeric DNA polymerase

a complex of two DNA polymerase holoenzymes that move as a unit during DNA replication.

termination sequences (ter sequences)

in E. coli, a pair of sequences in the chromosome that bind a protein known as the termination utilization substance (Tus), which stops the movement of the replication forks.

conditional mutants

Allow researchers to identify loss-of-function mutations in essential genes

temperature-sensitive mutants

a type of conditional mutant that displays a normal phenotype at the permissive
temperature, but a different phenotype at the nonpermissive (restrictive) temperature.

process to identify TS mutants

• Mutagenize cells
• Plate on growth media and grow at permissive temperature
• After colony formation, replica plate onto 2 plates
• Grow 1 plate at permissive temperature and the other plate at the
nonpermissive temperature
• Identify colonies that do not grow at nonpermissive temperature
• Use cells from master plate to identify nature of the mutation
(sequencing, phenotype, etc)

Because there are numerous essential genes, most of the ts mutants in this type of screen will…

…not affect the gene and/or process you are targetting.

enzymatic actions of DNAP

1. dNTP enters the catalytic site of DNAP and H-bonds to the
template strand according to the AT/GC bp rules
2. The 3’ OH on the last nucleotide of the growing daughter
strand reacts with the phosphate group of the incoming dNTP
3. The energy needed to form a covalent ester bond is provided
by the exergonic hydrolysis of a phosphoanhydride bond which
releases pyrophosphate and energy

net result of enzymatic action of DNAP

daughter strand extended by 1 nucleotide in the 5’ to 3’ direction.

Processivity

• DNAP III holoenzyme is a processive enzyme, it does not dissociate from the template strand after catalyzing the attachment of an incoming nucleotide
• The β subunit acts as a clamp that keeps DNAP attached to the template DNA, allowing it to catalyze the attachment of numerous successive nucleotides
• Without the β subunit, DNAP can add 20 nt/second and will dissociate after approximately 10 nucleotides have been added
• With the β subunit, DNAP can add 750 nt/second and has been shown to synthesize leading strands > 500,000 successive nucleotides.

DNAP lll has high fidelity, it only makes a ~ 1 mistake for every 100 million synthesized nucleotides. Three factors contribute to DNAP fidelity:

base pair stability, structure of the active site, and proofreading

Base pair stability

hydrogen bonds between correct base pairs much more stable (1 mistake per 1000 nt)

Structure of the active site

incorrect base pair distorts the double helix causing it to fit in the active site less well (1 mistake every 100,000 – 1 million nt).

proofreading function

the ability of DNA polymerase to remove mismatched bases from a newly made strand.