Topic 3 (Chapter 12.1, 12.2, 11.2)

two requirements of DNA replication

• accurate (errors can multiply fast during cell division)

• fast

three proposed models of DNA replication

• conservative

• dispersive

• semiconservative

conservative DNA replication

the entire double stranded DNA molecule serves as a template for an entire new molecule

• the original DNA molecule is fully conserved

dispersive DNA replication

both strands break down into template pieces and then are reassembled

• each resulting molecule contains interspersed fragments of old and new DNA

• none of the original molecule is completely conserved

semiconservative replication of DNA

each strand serves as a template for the synthesis of a new strand

• each original strand remains intact but are not combined in the same molecule

experiment that found semiconservative replication

tracked isotopes of nitrogen over generations of E coli cells. these cells were subjected to equilibrium density gradient centrifugation.

• light sample produced high band

• heavy sample produced low band

• both produced intermediate band (rules out conservative which expects two bands)

• added light two for second replication after intermediate band formed and saw two bands at intermediate and light which was also seen in all successive replications, with the light becoming progressively stronger (rules out dispersive

major steps of DNA replication

1. initiation

2. unwinding

3. elongation

4. termination

initiation in prokaryotes

initiator proteins bind to oriC and cause a short section of DNA to unwind which then allows helicase and other single stand binding proteins to attach to the strand

OriC

specific sequence on circular prokaryotic DNA that is the origin of replication

unwinding in prokaryotes

• one helicase binds to each single strand and moves from 5’ to 3’ breaking hydrogen bonds

• single stranded binding proteins stabilize the single stranded areas of the DNA

• DNA gyrase relieves strain ahead of the replication fork

single stranded binding proteins in prokaryotes

attach tightly to exposed single stranded DNA by forming tetramers. They can bind to any single stranded DNA and are not base dependent.

• protects single stranded DNA

• prevents secondary DNA structure

DNA gyrase

type II topoisomerase that controls the supercoiling of DNA

• makes a double-strand break in one segment of the DNA helix ahead of the replication fork to reduce the torsional strain that builds up ahead of the replication fork as a result of the unwinding

• once it is unwound, it releases the broken ends of the DNA using ATP, effectively removing a twist in the DNA

what does DNA polymerase need to attach to nucleotides

a pre-existing 3’-OH group

direction of DNA synthesis

5’ → 3’

primase

synthesizes short stretches of RNA nucleotides (primers) in order to provide a 3/-OH group to which DNA polymerases can attach nucleotides. Does this by forming a complex with helicase at the replication fork

• primase does not need an OH group to start because it is an RNA polymerase

location of primers

on the leading strand, it is only needed at the 5’ end of the newly synthesized strand. On the lagging strand, where replication is discontinuous, a new primer must be generated at the beginning of each okazaki fragment

function of DNA polymerase I

removes and replaces primers

function of DNA polymerase III

elongates DNA

DNA polymerase III

large multiprotein complex that synthesizes DNA in 5’ to 3’ direction. can efficiently and accurately synthesize new DNA molecules

• 5’-3’ polymerase activity allows it to add new nucleotides

• 3’-5’ exonuclease activity allows it to remove nucleotides in the 3’-5’ direction to correct errors

high processivity

the capability of adding many nucleotides to the growing DNA strand without releasing the template.

• DNA polymerase III has this property. it can continue to synthesize DNA until the template has been completely replicated

leading strand

DNA is synthesized towards the fork in one continuous DNA strand

lagging strand

DNA is synthesized away from the fork in discontinuous strands called okazaki fragments

DNA polymerase I

large multiprotein complex used to replace RNA primers and synthesize DNA in its place

• has 5’ to 3’ polymerase activity to synthesize DNA that replaces primers but with low processivity

• has 3’ to 5’ exonuclease activity which gets rid of the RNA primers and also for proofreading

DNA polymerase II, IV, V

not used in replication, for DNA repair

DNA ligase

forms a phosphodiester bond to fix the nick formed between the intial nucleotide added by DNA polymerase III and the final nucleotide added by DNA polymerase I

• essentially joining okazaki fragments together to make a continuous strand of DNA replication

two ways of ending replication in prokaryotes

• two replication forks meet

• termination sequence

How does termination by sequence work in prokaryotes

Tus, a termination protein binds to Ter sequence to create a Tus-Ter complex that blocks the movement of Helicase to prevent further DNA replication

• blocs a replication fork moving in one direction but not the other

Coordination of leading and lagging strand synthesis

• DNA polymerase III and I are bound at the replication fork and move together

• DNA polymerase III falls off lagging strand after completing an Okazaki fragment

Three methods for accuracy of DNA replication

1. Low error by polymerase

2. Proofreading using 3’-5’ exonuclease activity

3. Mismatch repair systems

Mismatch repair system

Process that occurs after replication with after abnormalities in DNA secondary structures occur and are recognized.

• enzymes that recognize this excise the incorrectly paired nucleotide and replace it

Prokaryotic theta replication

1. Double stranded DNA unwinds at the replication origin

2. Replication bubble forms with a replication fork at each end

3. Replication proceeds around the circle

4. Eventually two circular DNA molecules are formed

Prokaryotic rolling circle replication

1. Replication is initiated by a break in one of the strands

2. DNA synthesis begins at the end of the broken strand using the inner strand as a template, the 5’ end of broken strand slowly leaving circle

3. Broken strand is cleaves and becomes a single stranded linear DNA, leaving a double stranded circular DNA molecule behind

4. These linear strands might circular use and serve as a template for a complementary strand

Multiple circular DNA molecules are formed

Origin recognition complex (ORC)

Multi protein complex that binds to to specific sequences of DNA and initiates replication by loading Helicase onto the double stranded DNA

What phase is Helicase loaded onto double stranded DNA in eukaryotes

During G1 phase of the cell cycle

What phase is Helicase activated in eukaryotes

During S phase, it begins separating double strands

Topoisomerase in eukaryotes

Remove supercooling ahead of the replication fork by clamping tightly to the DNA and breaking its strand, then once relaxed, they resealable it

The DNA polymerases involved in eukaryotic replication

Alpha, delta, epsilon

DNA polymerase alpha

initiator of DNA synthesis and repair

• has primase activity to synthesize an RNA primer

• Has synthesis activity to synthesize a short string of nucleotides following primer

DNA polymerase delta

Synthesizes lagging strand

DNA polymerase epsilon

Synthesizes leading strand

Termination of DNA replication in eukaryotes

The forms run into each other

Challenges of eukaryotic replication

• very large genome

• Wrapped around histones

• Needs to be coordinated across chromosome

How is large eukaryotic DNA replicated fast

Thousands of origins of replications

How does replication work with multiple origins of replication

• at each origin, a replication bubble is formed

• Synthesis occurs on both strands at each end of every bubble so replication forms proceed outward

• The forks of adjacent bubbles run into each other and the segments fuse

Replicon

Unit of replication consisting of DNA independently replicated starting from one origin of replications

Packaging of eukaryotic DNA

Highly organized and condensed into nucleosomes when DNA is wrapped around histone proteins

How does replication happen with nucleosomes present

1. Replication fork disrupts original nucleosomes

2. Pre-existing histones are redistributed to new DNA

3. Newly synthesized histones are added to complete the formation of new nucleosomes (strands will have both new and old nucleosomes)