Chapter 11: DNA Replication

DNA replication relies on the __________ of DNA strands

DNA replication relies on the __________ of DNA strands

complementarity

Parental and daughter DNA segments are interspersed in both strands following replication

dispersive model

Meselson and Stahl Experiment

1. Add an access of 14N-containing compounds to the growth medium so all of the newly made DNA will contain 14N.

2. Incubate the cells for various lengths of time.

3. Lyse the cells by the addition of lysozyme and detergent, which disrupt the bacterial cell wall and cell membrane, respectively.

4. Load a sample of the lysate onto a CsCl gradient.

5. Centrifuge the gradients until the DNA molecules reach their equilibrium densities.

6. DNA within the gradient can be observed under a UV light.

what did Meselson and Stahl see after 0’ 20’ and 40’

0’ 15N/15N band

20’ 14N/15N band

40’ 14N/14N band and 14N/15N band

Synthesis of DNA proceeds __________ around the bacterial chromosome

bidirectionally

DNA Polymerase III

main replication enzyme, synthesizes in 5' to 3' direction

-Needs primer cuz it needs a free 3'OH end

-Antiparallel to template strand

-Gives rise to leading/lagging strand

removes RNA primers

DNA Polymerase I

single primer synthesized towards fork

leading strand

3' to 5', needs several primers away from fork as fork unwinds

lagging strand

The origin of replication in E. coli is termed _____

oriC

origin of Chromosomal replication

Three types of DNA sequences in oriC are functionally significant

• AT-rich region

• DnaA boxes

• GATC methylation sites

events that occur at OriC region

1. DnaA proteins bind to DnaA boxes and to each other

   1. Additional proteins bind to bend DNA

   2. Strands separate at AT-rich region

2. DnaB/helicase binds to origin

3. DnaC assists

4. Helicase separates DNA bidirectionally creating 2 replication forks

1. Composed of six subunits

2. Travels along the DNA in the 5’ to 3’ direction

3. Uses energy from ATP

DnaB / Helicase

__________ are involved in regulating replication

• Need to ensure only one round of replication

GATC methylation sites

______________ methylates the A on both strands

• Immediately after replication, the daughter strand is not methylated

• Takes several minutes to become methylated

Initiation of replication only occurs efficiently on fully methylated DNA

• Second round initiation is blocked

DNA adenine methyltransferase (Dam)

DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them

This generates _______ ahead of each replication fork, alleviated by______ through cutiing one of the strands ahead of the fork

positive supercoiling

Topoisomerase II also called DNA gyrase

_____________- bind to the separated DNA strands to keep them apart

• Bases are exposed and can hydrogen bond with individual nucleotides

Single-strand binding proteins

short RNA strands start, or prime, DNA synthesis made by primase are …..in length

10-12 nucleotides

criteria of DNA polymerase

1. 5' to 3'

2. Needs a free 3' OH (RNA primer provides it at the end)

3. New strands are antiparallel to template strand

DNA pol I and III involved in _______

DNA pol II, IV and V are….

Normal replication

.DNA repair and replication of damaged DNA

• Responsible for most of the DNA replication

• Composed of 10 different subunits

• The alpha subunit catalyzes PHOSPHODIESTER bond formation between adjacent nucleotides (DNA synthesis)

• The other 9 fulfill other functions

• The complex of all 10 subunits is referred to as the DNA pol III holoenzyme

DNA pol 3

• Composed of a single polypeptide

• Removes the RNA primers and replaces them with DNA

DNA pol 1

Subunit Composition of DNA polymerase III Holoenzyme

T/F: Bacterial DNA polymerases may vary in their subunit composition, HOWEVER ALL HAVE SIMILAR CATALYTIC SUBUNIT

true

how does DNA pol 1 remove RNA primers

It uses a 5’ to 3’ exonuclease activity to digest the RNA and 5’ to 3’ polymerase activity to replace it with DNA

_______ catalyzes the formation of a phosphodiester bond to connect DNA fragments

DNA ligase

DNA helicase and primase are physically bound to each other to form a complex called the ________

• This complex better coordinates the actions of helicase and primase

primosome

The primosome is physically associated with two DNA polymerase holoenzymes to form the __________

replisome

Two DNA pol III proteins act in concert to replicate both the leading and lagging strands

The two proteins form a_________ that moves as a unit toward the replication fork

dimeric DNA polymerase

Upon completion of an Okazaki fragment, the enzyme releases the lagging template strand

The________ then reloads the polymerase at the next RNA primer

And another LOOP is formed

clamp loader complex

On the opposite side of the chromosome to oriC is a pair of termination sequences called_______

ter sequences

The protein tus (________) binds to the ter sequences

tus bound to the ter sequences stops the movement of the replication forks

termination utilization substance

T1 prevents the advancement of the fork moving__________

T2 prevents the advancement of the fork that is moving __________

left to right (CCW)

right to left (CW)

• Intertwined circular molecules are termed _______

• These are separated by the action of _____

catenanes

topoisomerase 2

The dna mutants fell into two groups when shifted to the non-permissive temperature

Some showed a rapid arrest

• These genes encoded enzymes needed for replication of the DNA

Other mutants completed their current round of replication but could not start another

• Encoded genes needed for initiation of replication

DNA polymerases catalyzes the formation of a covalent (ester) bond between the

Innermost phosphate group of the incoming deoxyribonucleoside triphosphate and 3’-OH of the sugar of the previous deoxynucleotide

In the process, the last two phosphates of the incoming nucleotide are released in the form of

pyrophosphate (PPi )

DNA polymerase III remains attached to the template as it is synthesizing the daughter strand, this is a _______ feature

processive

β subunit forms a dimer in the shape of a ring around template DNA

• It is termed the clamp protein

• Once bound, the β subunits can freely slide along______

• Promotes association of holoenzyme with DNA

dsDNA

what 5 subunits make up the clamp loader?

τ, γ, δ, δ′, ψ, and χ

In the absence of the β subunit

• DNA pol III falls off the DNA template after about 10 nucleotides have been polymerized

• Its rate is ___________

In the presence of the β subunit

• DNA pol III stays on the DNA template long enough to polymerize up to 500,000 nucleotides

• Its rate is ______________

~ 20 nucleotides per second

~ 750 nucleotides per second

reasons why fidelity is high

• Stability of base pairing

• Structure of the DNA polymerase active site

• Proofreading function of DNA polymerase

Configuration of the DNA polymerase active site

• Helix distortion caused by mispairing prevents incorrect nucleotide fitting properly in active site

known as….

induced-fit phenomenon

• DNA polymerases use a____________ activity to digest the newly made strand until the mismatched nucleotide is removed

• DNA synthesis then resumes in the 5’ to 3’ direction

3’ to 5’ exonuclease

the only DNA pol with 5’ to 3’ exonuclease activity

DNA pol 1

Evidence for Multiple Origins of Replication

A pulse of radiolabeled nucleoside was taken up by cells and incorporated into newly made DNA strands.

Labeled areas were interspersed between non-labeled, indicating multiple regions had initiated replication

Origins of replication in Saccharomyces cerevisiae are termed ARS elements (………..)

• They are about 50 bp in length

• They have a high percentage of A and T

• ARS consensus sequence (ACS)

Autonomously Replicating Sequence

ACS

A T T T A T (A or G) T T T A

Eukaryotic Replication begins with assembly of the ____________

prereplication complex (preRC)

components of preRC

Origin recognition complex (ORC)

MCM Helicase

--Binding of MCM completes DNA replication licensing

Cdc6

Cdc1

six-subunit complex that acts as the first initiator of eukaryotic DNA replication

ORC

eukaryotic replication steps

1. during G1 phase ORC binds to origin

2. Cdc6, Cdc1, & MCM helicase assemble onto origin to form preRC; MCM helicase binds to leading strand

   1. during S phase, preRC is converted to an active replication site by phosphorylation via protein kinases ; Cdc6, Cdc1, and ORC are released, MCM helicase moves in 3’ to 5’ direction and DNA rep. proceeds bidirectionally Binding of MCM completes DNA replication licensing

mammalian DNA polymerases (total over 12)

alpha (α), delta (δ), epsilon (ε) and gamma (γ) have the primary function of replicating DNA

α, δ and ε →

γ →

NUCLEAR DNA

MITOCHONRIAL DNA

______ is the only polymerase to associate with primase

DNA pol alpha

The DNA pol α/primase complex synthesizes a short RNA-DNA hybrid primer

10 RNA nucleotides followed by 20 to 30 DNA nucleotides

The exchange of DNA pol α for ε or δ is required for elongation of the leading and lagging strands. This is called _____________

polymerase switch

• DNA pol ε is used for the processive elongation of the --------

• DNA pol δ is used for the ----------

leading strand

lagging strands

DNA pol β is not involved in DNA replication

It plays a role in removal of incorrect bases from damaged DNA

• Involved in the replication of damaged DNA

• They can synthesize a complementary strand over the abnormal region

translesion-replicating polymerases

Distinct from prokaryotic replication ________- runs into primer of adjacent Okazaki fragment

• Pushes portion of primer into short flap

• ___________ removes the primer

Polymerase δ

Flap endonuclease

Long flaps are removed by __________--

DNA2 nuclease/helicase

Telomeric sequences consist of moderately repetitive tandem arrays

• Several guanine nucleotides

• Many thymine nucleotides

________ that is 12-16 nucleotides long

3’ overhang

At the_____- of linear chromosomes - the end of the strand cannot be replicated!

3’ ends

Telomerase contains protein and RNA…..

This allows the telomerase to bind to the 3’ overhang

template

The RNA is complementary to the DNA sequence found in the telomeric repeat

enzymatic action of telomerase

Step 1: binding

--telomerase binds to 3’ overhang

Step 2: polymerization

--telomerase synthesizes 6-nucleotide repeat

Step 3: translocation

--telomerase moves 6 nucleotides to the R and makes another repeat

complementary strand is made by primase, DNA poly., ligase

These steps are repeated many times to lengthen one strand

Cells become _________ when telomeres are short

senescent