Requirements for the Genetic Material

• 1. Replication

Reproduced and transmitted faithfully from cell to cell-generation

to generation.

• 2. Information Storage

Biologically useful information in a stable form.

• 3. Expression of Information

Express itself: Other biologically important molecules, and

ultimately cells and organisms, will be produced and maintained.

• 4. Variation (by mutation)

Capable of variation: some change is required

REPLICATION

OF DNA

Replication of DNA

• What is the mode of DNA

replication and how was

that determined?

Fig. 10-1

Replication of DNA: 3 possible models

Fig. 10-2

Meselson-Stahl Experiment

• Grew E. coli in medium containing ammonium chloride (15NH4Cl) as sole N source – 15N = “heavy” but nonradioactive isotope

– After multiple generations, all N-bases of DNA contain 15N

– Extracted DNA and centrifuged in density gradient (CsCl)

• Grew 15N labelled cells in regular medium (14NH4Cl) – Collected samples after 1st, 2nd and 3rd generations

– Extracted DNA and centrifuged in density gradient (CsCl)

• Compared banding patterns observed in density gradients

Meselson-Stahl Experiment

Fig. 10-3

In the Meselson-Stahl experiment, if DNA is

replicated conservatively, then after 2

generations of replication there would be…

• A) one “old” band and one “new” band

• B) one “hybrid” band

• C) one “hybrid” band and one “new” band

• D) one “hybrid” band and one “old” band

• E) one “old” band, one “hybrid” band, and one “new” band

DNA Replication • Bacterial & eukaryotic models

– Similar process:

• Initiation

• Elongation

• Termination

– Differences due to differences in DNA

structure and complexity of DNA coiling

• Circular vs. linear DNA

• Single vs. multiple origins of replication

• Absence or presence of nucleosomes

DNA Replication in Bacteria

Fig. 10-6

DNA Replication

• Unwind & denature double helix – Helicases

• Unwind, open & stabilize helix

• DnaA, DnaB, DnaC

– Stabilized by SSB’s (single-stranded binding proteins)

– Problem • Unwinding creates

supercoiling causes torsional stress

• Relieved by DNA gyrase

• SS or DS nicks

Fig. 10-9

DNA Replication

• Initiate synthesis

– RNA primase (RNA polymerase) adds an

RNA primer

• ~5-15 nucleotides

Fig. 10-9

The role of the DnaA protein in bacterial DNA

replication is to…

• A) prevent renaturation of the two DNA strands during replication

• B) synthesize and RNA primer

• C) relieve the tension of supercoiling

• D) initially unwind the DNA double helix

• E) detect replication errors

DNA Replication

• Chain elongation

– DNA pol III

• Requires free 3’-OH to bind

• Adds nucleotides 5’ 3’

Fig. 10-9

Fig. 10-7

Elongation

• Elongation is...

– Simultaneous

• Both strands at once

– Bidirectional

• Strands are antiparallel

– Continuous & discontinuous

processes

• Leading strand (continuous)

• Lagging strand (discontinuous

– Okazaki fragments

Fig. 10-11

simultaneous synthesis

Okazaki fragments are a consequence of…

• A) the inability of DNA polymerase to initiate a new DNA strand

• B) the inability of DNA polymerase to correct replication errors

• C) random strand breakage due to supercoiling

• D) mutations in the gene for DNA ligase

• E) the inability of the DNA polymerase to synthesize in the 3’-5’ direction

DNA Replication

• Primers removed and lagging strand gaps

filled

– DNA pol I

• Cleaves out RNA primer and replaces with DNA

• Has both 5’ 3’ exonuclease and polymerase

activities

– DNA ligase

• Forms final phosphodiester bond to fill gap

Chain Elongation

• Enabling of concurrent synthesis

– DNA pol III is a dimer

– Loop forms to keep antiparallel template

strands effectively 3’ 5’ relative to DNA pol III

• 1000-2000 bp

Fig. 10-12

Proofreading

• Critical that new strand is exact

complement of template

• DNA pol I & III have 3’ 5’ exonuclease

activity

– Recognition & replacement of mismatches

during elongation

– “back-up” and replace mismatched bases

• DNA pol II

– Active in DNA repair to external damage

(i.e., UV light)

Polymerase Comparison

Bacterial DNA Replication

Summary

Fig. 10-13 dna replication

All known bacterial DNA polymerases…

• A) can initiate DNA chain synthesis

• B) have 5’ to 3’ polymerization activity

• C) have 5’ to 3’ exonuclease activity

• D) have 3’ to 5’ polymerization activity

• E) all of the above

Replication of Eukaryotic DNA

• Problems

– Larger genomes

– Eukaryotic DNA pols are slower

– Nucleosomes

– Linear chromosomes

Replication of Eukaryotic DNA

• Solutions

– Multiple origins of replication

• Yeast : 250-400 replicons

• Mammals: up to 25,000 replicons

– More types (14) of polymerases

• Different activities / operate under different conditions

– More polymerase molecules per cell

• E. coli ~400/cell

• Homo sapiens ~50,000/cell

Comparison of Replication

Rates

• E. coli 4.7 kb ~20-40 min

• Drosophila 120 kb ~ 3 min

• Homo sapiens 3300 kb ~ 7 hrs

Replication of Eukaryotic DNA

• Replication problems at

ends (telomeres) of linear

chromosomes

– RNA primer at terminal end

• Once removed, no 3’-OH

available for addition of DNA

nucleotides

• Can lead to telomere

shortening (cellular clock)

– Fetal tissue culture cells - 60-

80 divisions max.

– Adult cells - 10-20 divisions

max Fig. 10-16

Telomeres

• Repeated sequences

– Tetrahymena – a protozoan

• TTGGGG tandem repeats

• Overhang on G-rich strand of 12-16 bases

• G-quartets – form loops on ends of chromosomes

Fig. 10-17

Telomerase

• A ribonucleoprotein – Contains RNA (-AACCCCAAC-)

• Recognizes telomeric sequence and adds repeats – RNA primase

– DNA pol I & ligase

• Typically low activity in somatic cells; high activity in cancerous cells

Fig. 10-17

Telomerase…

• A) is a ribonucleoprotein

• B) has reverse transcriptase activity

• C) adds short tandem repeats to the ends of chromosomes during DNA replication

• D) functions to replicate DNA at the ends of linear chromosomes

• E) all of the above

Recombination (Crossing Over)

1. Homologs pair and synapses form

2. Endonucleases “nick” DNA at adjacent sites on both homologs – SS

3. Ends are displaced and pair with homologous sequence on other duplex

Fig. 10-18

Recombination

4. Ligase seals nicks

– Creates heteroduplex

5. Branches migrate

– H-bonds “unzip” and form

complementary bonds

with other duplex

6. Duplexes separate

& rotate

– Chiasmata

Fig. 10-18

Recombination

7. Endonucleases nick

opposite sides of

chi form

8. Homologs are ligated

and separated

Fig. 10-18

Recombination…

• A) is a ribonucleoprotein

• B) has reverse transcriptase activity

• C) adds short tandem repeats to the ends of chromosomes during DNA replication

• D) functions to replicate DNA at the ends of linear chromosomes

• E) all of the above

Gene Conversion

• Mismatch after crossover can result in

mutation

Fig. 10-19