genetics ch 11

DNA replication

the process by which the genetic material is copied

How are the original DNA strands used in DNA replication

as templates for the synthesis of new strands

DNA replication relies on

the complementarity of DNA strands (the AT/GC rule or Chargaff’s rule)

Process of DNA replication

- The two DNA strands come apart

- Each serve as a template strand for the synthesis of new strands

- The two newly made strands = daughter strands

- The two original ones = parental strands

How is Hydrogen bonding regarding Chargaff’s rule

hydrogen bonding between individual nucleotides and the template strands must obey the AT/GC rule

The three possible mechanisms for DNA replication

Conservative model, semiconservative model, dispersive model

Conservative model

both parental (template) strands stay together after DNA replication

Semiconservative model

the double-stranded DNA contains one parental and one daughter strand following replication

Dispersive model

parental and daughter DNA are interspersed in both strands following replication

Matthew Meselsona and Franklin Stahl findings

found a way to experimentally distinguish between daughter and parental strands

What was the purpose of Matthew Meselsona and Franklin Stahl’s experiment

to devise a method to investigate the three models of DNA replication

Meselson and Stahl experiment

- Grow E. coli in the presence of 15N (a heavy isotope of Nitrogen) for many generations

- The population of cells had heavy-labeled DNA

- Switch E. coli to medium containing only 14N (a light isotope of Nitrogen)

- Collect sample of cells after various times

- Analyze the density of the DNA by centrifugation using a CsCl gradient

Meselson and Stahl hypothesis

This experiment aims to determine which of the three models of DNA replication is correct

Where does DNA synthesis begin

the origin of replication (each bacterial chromosome only has one)

What direction does DNA synthesis move

Synthesis of DNA proceeds bidirectionally around the bacterial chromosome (producing two replication forks)

What ends bacterial DNA replication

the replication forks eventually meet at the opposite side of the bacterial chromosome

how do replication forks move

in the opposite direction from the origin

what is the replication fork

the site where the parental strands have separated and new daughter strands are being made

What is the origin of replication in E. coli called

oriC

what is oriC

origin of chromosomal replication

What are the three types of DNA sequences in oriC that are functionally significant

AT-rich region, DnaA boxes, GATC methylation sites

What initiates DNA replication

the binding of DnaA proteins to the DnaA box seqquence

What does the binding of DnaA proteins/DnaA box sequences stimulate

the cooperative binding of an additional 20-40 DnaA proteins to form a large complex

What happens when the 20-40 DnaA proteins that form a large complex

the region wraps around the DnaA proteins and separates the AT-rich region

What is helicase composed of

six subunits

how does helicase travel

along the DNA in 5’ to 3’ direction, using energy from ATP

What does DNA helicase do

breaks the hydrogen bonds between the two DNA strands, generating two single strands (replication forks)

What does DNA helicase’s separation of hydrogen bonds cause

it generates positive supercoiling ahead of replication fork

DNA gyrase (topoisomerase II)

travels ahead of the helicase and alleviates the supercoils helicase produces

What do single-strand binding proteins do

bind to the separated DNA strands to keep them apart

What does DNA primase do

synthesizes short (10-12 nucleotides) RNA primers

What do the RNA primers produced by DNA primase do

start (prime) DNA synthesis

RNA primers on leading and lagging strands

the leading strand has a single primer; the lagging strand needs multiple primers (they are later removed and replaced with DNA)

What are DNA polymerases

enzymes that catalyze the attachment of nucleotides to make new DNA

The five proteins with polymerase activity in E. coli

DNA pol I, II, III IV, and V

DNA pol I and III

normal DNA replication

DNA pol II, IV, and V

DNA repair and replication of damaged DNA

DNA pol I composition

composed of a single polypeptide

DNA pol I role

removes the RNA primers and replaces them with DNA

DNA pol III composition

10 different subunits

DNA pol III role

responsible for most of the DNA replication

DNA pol III holoenzyme

what the complex of all 10 DNA pol III enzymes are reffered to as

What are the 10 different subunits of DNA pol III

the alpha subunit and nine others

What are the specific functions of the 10 DNA pol III subunits

Alpha subunit synthesizes DNA, while the other 9 have different functions

How is the leading strand synthesized (RNA primers)

one RNA primer is made at the origin

How is the leading strand synthesized (DNA pol III)

DNA pol III attaches nucleotides in a 5’ to 3’ direction as it slides toward the opening of the replication fork

What is required for the lagging strand to be synthesized (in relation to the RNA primers)

Many RNA primers are required (RNA primers repeated initiate the synthesis of short fragments of DNA)

How is the lagging strand synthesized (DNA pol III)

DNA pol III uses the RNA primers to synthesize small DNA fragments (1000 to 2000 nucleotides each) away from the fork

How is the lagging strand synthesized (direction)

Synthesizes in the 5’ to 3’ direction, but it occurs away from the replication fork

what does DNA pol I remove

the RNA primers and fills the resulting gap with DNA

how does DNA pol I remove the RNA primers and add DNA

it uses the 5’ to 3’ exonuclease activity to digest the RNA, 5’ to 3’ polymerase activity to replace it with DNA

What is missing after DNA pol I replaces the RNA primers with DNA

a covalent bond

DNA ligase

catalyzes a phosphodiester bond, thereby connecting the DNA fragments

How does DNA polymerase work in relation to the innermost phosphate and the ‘OH

it catalyzes a phosphodiester bond between the innermost phosphate group of the incoming deoxynucleotide triphosphate and the 3 ‘OH of the sugar of the previous deoxynucleotide

pyrophosphate

the last two phosphates of the incoming nucleotide in the reaction of DNA polymerase are released

What is DNA polymerase III

a processive enzyme

How fast does DNA synthesis occur

750 nucleotides/second

Why is DNA synthesis able to occur so fast

because DNA polymerase III remains attached to the template as it’s synthesizing the daughter strand

Why does DNA polymerase have a processive feature

it’s due to several different subunits in the DNA pol III holoenzyme

the processive subunits of DNA polymerase III

beta subunit- shaped like a ring, called clamp protein

gamma subunit- needed for beta to initially clamp onto the DNA, called the clamp-loader protein

What happens in DNA polymerase III in the absences of the beta subunit

DNA pol III falls off the DNA template after a few dozen nucleotides have been polymerized, ~20 nucleotides per second

What happens in DNA polymerase III in the presence of the beta subunit

DNA pol III stays on the DNA template long enough to polymerize up to 50,000 nucleotides, ~75 nucleotides per second

Where us the termination sequence located

opposite of the oriC

What is the termination sequence called

ter sequences, T1 and T2

What is tus

a protein termed termination utilization substance

what does the protein tus do

binds to the termination sequences and stops the replication forks from moving past the ter sequences

What does T1 do

prevents advancement of fork from left to right

What does T2 do

prevents advancement of fork from right to left

When does DNA replication end

when oppositely advancing forks meet (usually at T1 or T2)

How does DNA ligase work during the termination of replication

it covalently links the two daughter strands, creating two circular double stranded DNA molecules

DNA replication often results in

two intertwined molecules

what are intertwined circular molecules termed

catenanes

How are catenanes separated

they are separated by the action of topoisomerases

How do topoisomerases separate catenanes

DNA topoisomerases catalyze catenanes which temporarily break into the DNA strands and then rejoin them after the strands have become unlocked

What is DNA replications degree of fidelity

it has a high degree of fidelity, so mistakes during the process are extremely rare

How many mistakes does DNA pol III make

_{only one mistake per 10}8 _{bases are made}

Why is the fidelity of DNA replication so high

- instability of mismatched pairs

- configuration of the DNA polymerase active site

- proofreading function of DNA polymerase

instability of mismatched pairs

complementary base pairs have much higher stability than mismatched pairs, this feature only accounts for part of the fidelity (it has an error rate of 1 per 1000 nucleotides)

configuration of the DNA polymerase active site

DNA polymerase is unlikely to catalyze bond formation between mismatched pairs, this induced-fit phenomenon decreases the error rate to a range of 1 in 100,000 to 1 million

proofreading function of DNA polymerase

- DNA polymerases can identify a mismatched nucleotide and remove it from the daughter strand

- the enzyme uses 3’ to 5’ exonuclease activity to remove the incorrect nucleotide

- it then changes direction and resumes DNA synthesis in the 5’ to 3’ direction

Eukaryotic DNA replication

it’s not as understood as bacterial replication and it’s more complex

Eukaryotic DNA replication characteristics

large linear chromosomes, tight packaging within nucleosomes

What kind of chromosomes do Eukaryotes have

long linear chromosomes

What do Eukaryotes long linear chromosomes require, and why

they require multiple origins of replication to ensure that DNA can be replicated in a reasonable time

What did Huberman and Riggs provide evidence for

multiple origins of replication

What direction is DNA replication in Eukaryotes

replication proceeds bidirectionally from many origins of replication

What are Eukaryotic origins of replication in Saccharomyces cerevisiae called

ARS elements

What does ARS elements stand for

Autonomously Replicating Sequence

Why are Autonomously Replicating Sequences necessary

for initiating chromosome replication

Characteristics of Autonomously Replicating Sequence

- They have high percentages of A and T

- they have three or four copies of an ARS consensus specific sequence (similar to the bacterial DnaA boxes)

Where does DNA replication in Eukaryotes begin

begins with assembly of the prereplication complex (preRC) during the G1 phase of the cell cycle

Important part of DNA replication in eukaryotes

Origin recognition complex (ORC)

What is the ORC

a six-subunit complex that acts as the initiator of eukaryotic DNA replication by binding to ARS element (G1 phase), other preRC proteins bind (including MCM helicase)

How does MCM helicase work in eukaryotic DNA replication

the binding of MCM helicase completes the assembly of the prereplication complex, which gets activated via phosphorylation

What are the different Eukaryotic DNA polymerases

alpha, delta, epsilon, and gamma have the primary function of replicating DNA (alpha, delta, and epsilon are nuclear DNA)

How many polymerases does eukaryotic DNA contain

more than a dozen

What polymerase associates with primase

DNA pol alpha

How does DNA pol alpha associate with primase

the DNA pol alpha/primase complex synthesizes a short RNA-DNA hybrid (10 RNA nucleotides followed by 20-30 DNA nucleotides)

What is the polymerase switch

the exchange of DNA pol alpha for gamma or epsilon