7/24/01

Lecture #12 - DNA replication

Why bother studying DNA replication?

1) Fidelity. It's important for replication to have high fidelity. Mistakes in replicating DNA cause mutations that can be transferred from one generation to the next. It needs to be accurate!
2) Regulation. Eukaryotes have multiple origins of replications and multiple chromosomes. They need to replicate all the DNA, but only once - need tight control. Tied to the cell cycle control
3) Machinery is impressive

5 Basic Facts of DNA Replication

1) Terminology

ORIGIN - where replication initiates

TERMINUS - where replication finishes

UNIDIRECTIONAL - replication proceeds in 1 direction from the origin

BIDIRECTIONAL - replication forks go in both directions

(there are examples of both in nature)

Fig. 12.2

REPLICON (a favorite in Lewin) - unit of DNA synthesis which begins at the origin of replication and finishes at the terminus

Different types of organisms replicate in different ways

E. coli

genome contains 4 x 10⁶ b.p.
Circular chromosome
Single origin of replication (245 b.p.), an AT rich region
Single replicon
Bidirectional replication starts from the origin. When the forks run into each other, that's the end.
However, replication forks can be delayed, so the cell possesses a backup mechanism that ensures replication termination: The termination sites lie next to the halfway point. The L site lies slightly to the right of the halfway point and the R site lies slightly to the left of the halfway point. Termination sequences are directional - replication of R stops only at the R terminus and L stops only at the L terminus. The replication forks will only go so far before falling off. See Fig 12.7

Eukaryotic Cells

Linear chromosome
Multiple origins of replication (therefore think about regulation - must initiate from each origin once and only once per cell cycle)
Bidirectional

Relationship between transcription and DNA replication

both go on simultaneously
Work at different rates - replication (800-1000 nuc/sec) is more rapid than transcription (RNAP transcribes 40 nuc/sec)
In E.coli it is observed that the most highly transcribed genes/operons are transcribed in the same direction as the movement of the replication fork
Those transcribed in the opposite direction are those not highly expressed.
Inverting regions of the E. coli chromosome that contain highly transcribed genes so they now point in the opposite direction is lethal (presumably because they can't be transcribed at a high enough rate to keep the cell alive)

Replication machinery and RNAP - what happens when they encounter one another?

Same direction- DNA polymerase skips over RNAP (presumably - rather inexplicabel mechanism)
Opposite direction - DNA polymerase knocks RNAP off template

2) DNA polymerase is the enzyme that carries out DNA synthesis

Always synthesizes in the 5' to 3' direction
Reaction: DNA + dNTP (single nucleotides) --> DNA+1 + P-P (diphosphate)
(similar reaction as RNAP in carrying out RNA synthesis)

DNAP:

requires a primer (unlike RNAP)
can't initiate DNA synthesis de novo (without a primer)
primer must have -OH on 3' end
primer can be DNA/RNA
requires a template (something to copy)
normally complementary to the region to be synthesized

Activities of DNAP

1) polymerase 5'--> 3'
2) 3' -->5' exonuclease ("proof-reading")
3) 5'--> 3' exonuclease (Function: to remove primers from DNA, which are often RNA)

Fig. 13.1 - shows reaction DNA + dNTP --> DNA n+1 + P-P

Fig. 13.2 - 3' --> 5' proofreading If DNAP makes a mistake (incorporates wrong nucleotide), realizes the problem, backs up 1, cleaves the phosphodiester bond and gets a 2nd chance to insert the correct nucleotide, thereby decreasing the chance for error

3 DNA polymerases in E.coli

	DNA pol I	DNA Pol II	DNA Pol III
5' -- 3' pol	+	+	+
3' --> 5' exo	+	+	+
5' --> 3' exo	+	-	-
# subunits	1	1	10
gene	polA	polB	polC
function	DNA repair RNA pimer removal	DNA repair	major replicating polymerase can initiate at origin of replication

DNA polymerase I - 103 kD protein

Digest with protease - trypsin - digested into 2 pieces that have separate activities
68 kD - large fragment - 5' --> 3' pol, 3' --> 5' exo, lacks 5' --> 3' exo
35 kD - small fragment (no activity - ignore)
The large fragment is called the Klenow fragment, which is used in labs

3) Fidelity - ability to not make many mistakes

2 reasons why so accurate

1) presynthetic - Selects the right dNTP to match template strand / determines the correct base pair the incoming dNTP and template
2) "Proofreading" - 3 --> 5' exonuclease. If makes a mistake, it has a chance to correct it. Usually gets it right on the 2nd chance.

How often are mistakes made?

See chart on handout (Table 15.1 from Genes VI) Bottom line: not Often!

Reverse transcriptase

RNA dependent DNA polymerase. Discovered in retroviruses, e.g. AIDS
"error-prone" enzyme - introduces mutations at a higher rate (partially explains why AIDS can evade the immune system)

Taq polymerase

Used in PCR
No proofreading
"error-prone"
mistakes - 1/20,000 b.p.
number of mistakes can be increased by adding manganese, or changing the concentrations of the dNTPs

Other Thermostable DNA polymerases

Can use for PCR
Possesses proofreading ability -error rate in the 1/10^-5 - 10^-6 - 20x lower than Taq
Vent, Pfu (companies' names)
Better for PCR without mistakes

4) DNA replication is semi-conservative

one strand of the DNA serves as the template for the other
formal proof 1957-8: Meselson-Stahl experiment (heavy/light nitrogen - look at subsequent distribution in future generations)

5) DNA replication semi-discontinuous

one strand is synthesized in the 5 --> 3' direction continuously - "leading strand"
other strand is synthesized discontinuously - "lagging strand"
Synthesis of the lagging strands occurs as a series of smaller pieces called Okazaki fragments

Fig. 13.7

Semi-discontinuous nature
Place where unwound is called the replication fork
The leading strand moves in the same direction as the fork
The lagging strand is synthesized in the opposite direction than that of the fork (synthesized as Okasaki fragments)

Fig. 13.2

The leading strand is primed once
In the lagging strand, primers are initiated periodically
As the fork opens up, another primer can initiate another Okazaki fragment

Fig. 13.8

same information as Fig. 13.2 , but more complex
gaps exist between the fragments because a phosphodiester bond can't be made to the 5' end of the primer
aAlso, the RNA primer must be removed
DNA Polymerase I: The 5'--> 3' exonuclease activity recognizes the nick and degrades the primer
The DNA polymerase activity synthesizes DNA to replace the RNA
DNA Ligase seals the nick

Studies on DNA replication

Arthur Kornberg at Stanford - won the Nobel Prize in 1959
Studied DNA polymerase in E.coli. Worked up to the present - now is close to 90 yrs. old!
Genetics and Biochemistry*. Used both, but primarily a biochemist

1) Genetic

Make mutations - conditional mutants (expressed under one condition but not under another)
Temperature sensitive - defective in DNA replication
Permissive - low 37° C - functional
Restrictive - high 42° C - not functional
Isolated mutants - "dna" mutants in E.coli
2 classes
1) "quick stop" mutants - when the temperature was shifted from 37°to 42° (restrictive), it was observed that DNA replication stopped immediately. Postulated that the mutations were in the elongation machinery (that carries out DNA synthesis) - possible mutations in the DNA polymerase or in the supplied essential precursors (dNTPs)

2) "slow stop" - Temperature was again shifted to the restrictive temperature. Observation - able to finish replication but unable to initiate another round of DNA replication. Postulated that the mutants were defective in components that are involved in initiation - functioning in or around the origin of replication

2) Biochemical - in vitro experiments (DNA replication in test tubes)

Template DNA containing origin + dNTP + wt protein extract --> replication
Template + dNTP + mutant extract (dna^-)--> no replication
Template + mutant extract + purified proteins from wt extract--> replication
in vitro complementation - correlation between defect in mutant with loss of particular protein
Correlated defect present in DNA mutants with a particular protein
Purified protein could complement defect - in vitro complementation

Kornberg purified all the proteins required for replication in E.coli

Other enzymes (at replication fork; help with DNA synthesis)

1) DNA gyrase/topoisomerase

unwinds DNA at replication fork
relieves torsional stress but cutting strands and religating them

2) helicases

unwind DNA
don't cut strands of DNA - pull apart without cutting bonds
ATP requiring enzymes (need energy)
different helicases: DNA B, rep

3) Primases

Synthesize RNA primers
Categorize as DNA dependent RNAPs
Specific - synthesize RNA only at replication fork
Able to synthesize de novo (primase doesn't require a primer)
Primers are generally short (8-50 bonds), 1-2 kb apart on lagging strand
Primase in E.coli - product of dnaG gene

4) SSB - Single Strand Binding Protein

binds to single strand DNA cooperatively and stoichiometrically
functions at replication fork to prevent DNA from reforming duplex

5) DNA polymerase I

removes RNA primers (because it has 5'Æ3' exonuclease activity)

6) DNA ligase

seals nicks in DNA present in Okasaki fragments even after the RNA is removed

Fig. 13.3

5'--> 3' exonuclease "nick translation"
DNAP I recognizes the region containing 3'-OH end of the primer and removes it (whether DNA/RNA) and resynthesizes a new DNA (using 5' --> 3' polymerizing activity

Fig. 13.9

DNA ligase seals nicks
Using ATP or NAD as an intermediate, it catalyzes the formation of a phosphodiester bond

How does this all come together to synthesize DNA at the replication fork?

Take home message: The leading and lagging strands in E.coli and Eukaryotes are replicated by a single enzyme complex

Fig. 13.15

Large protein complex

The lagging strand forms a loop

DNA polymerase III - does the replicating

10 subunits , 4 major components

Component
# proteins
# / fork

Core: - 5' --> 3 pol

3' --> 5' exo
3
2

Linker - Tau

(holds complex together)
1
2

sliding clamp - ß
2 (homodimer)
2

clamp loader / unloader

gamma complex
5
1

(only lagging)

Sliding Clamp -ß

"clamps" DNA polymerase core enzyme to DNA
Its presence increase the rate (# bp/min) and the processivity (measures # bp synthesized before the enzyme falls off the template)
Based on in vitro experiments, in the absence of the clamp - DNA synthesis rate is 10 bases/sec. Processivity is 10 bases (on average) before it falls off
In the presence of the clamp - rate is 750 bases/sec and processivity is 50,000 bases before falls off
ß-subunit is very important for both rates!

Fig. 13.14

Structure of ß-sliding clamp
Donut shaped homodimer
Wraps around DNA and holds the polymerase to the DNA
In its absence, the polymerase is not strongly held to the DNA
The structure confirms experimental results

Clamp loader/clamp unloader complex

loads/unloads the ß-clamp on/off the lagging strand
Paradox: Want DNA synthesis to be rapid/processive (largely dependent on ß-clamp) yet for DNA synthesis on lagging strand, something is required to take it off.
How is it that the lagging strand is rapid and processive with the ß-clamp yet discontinuous?, i.e. it needs to associate, dissociate, then reasssociate somewhere else.
Also, how is the replication of both the leading and lagging strands done by a single enzyme complex?

Fig.13.15

Present model for DNA replication

Picture on the top of page 2 of the handout
Leading strand - initiated and continuous
Lagging strand - series of primers
Page 3 of the handout
1 complex - looping model - lagging strand forms a loop
As the primer moves down, the loop gets bigger
Once the fragment has been transcribed, the clamp unloader/loader dissociates at the end of the Okazaki fragment and attaches to the 3' end of the next RNA primer, so the polymerase III and the clamp are positioned to replicate a new fragment. New RNA primers are being made by the primase
Picture on bottom of page 2 of the handout - look only at the lagging strand
ß-clamp - associated either with clamp loader or with core polymerase III but not both
All happens very rapidly - 1/sec

Fig. 13.16 - can look at, but might be confusing

Fig. 13.18

Eukaryotic cells - same types of components (different names), not as well studied