DNA ReplicationLecture 11Fall 2008

• Read pgs. 305-312

Nucleic Acid Structure

• Deoxyribonucleic Acid (DNA)– Double strand

• Ribonucleic Acid (RNA)– Single strand

• Nucleic Acid: long chain of nucleotides

3 components of nucleotides• 5 carbon sugar• Phosphate group (PO4

-)• Nitrogenous bases

– Nucleoside = sugar + base (no phosphate group)

1

Fig. 5.27

Nucleic Acid Structure

Nitrogenous bases• Pyrimidines

• 6-membered ring of carbon and nitrogen

– Cytosine (C)– Thymine (T)– Uracil (U) replaces T in

RNA

• Purines• 6-membered ring fused

to a 5-membered ring

– Adenine (A)– Guanine (G)

2

Fig. 5.27

Nucleic Acid Structure

• 5 carbon sugar– Ribose in RNA– Deoxyribose in DNA

• Missing oxygen at 2’

• Carbons numbered 1 to 5 – prime’

3

DNA Structure

• Long chain of nucleotides– Allows for unique

arrangement of 4 bases

• Sugar-phosphate backbone– Phosphodiester linkage

• Covalent bond between sugar group of one nucleotide and phosphate group of another nucleotide

• 5’ end with phosphate group

• 3’ end with hydroxyl group (OH)

Fig. 5.27

4

DNA StructureDNA molecule• Double helix

– Two strands

• Antiparallel– Strands oriented

in opposite directions

• Complementary base pairing– T + A– C + G

• Hydrogen bonds between the base pairs

• Van der Waals interactions between stacked bases

Fig. 16.27

5

DNA Replication

• Cell division requires the duplication of genetic material

• DNA is a template– Two strands separate– Free nucleotides bond to

template and form “daughter” DNA strand

6

See Fig. 5.28

DNA Replication

• Origins of replications– Short stretches of DNA with

specific nucleotide sequence

– DNA separates, forming replication bubble

– Replication continues in both directions until completed

– Prokaryotes• One origin of replication

See Fig. 16.12

7

DNA Replication

• Origins of replications– Eukaryotes

• Many origins (100s to 1000s)• Replication bubbles eventually fuse

– Replication fork• Y-shaped region where parental DNA strand is unwound

into 2 single strands

Fig. 16.12

8

DNA ReplicationHow does DNA separate?• Helicases

– Unwinds and separates DNA strands– Catalyzes breaking of hydrogen bonds between nucleotides

• Single-strand binding proteins– Stabilizes separated strands

• Topoisomerase– Releases strain on

unwinding DNA – Cuts, twists and rejoins

DNA downstream of replication fork

Fig. 16.13

9

DNA Replication• How is DNA synthesis initiated?

– Primase• Adds a primer - short section of RNA

– 5-10 nucleotides long

• Necessary because DNA polymerases can only add nucleotides to an existing chain

Fig. 16.13

10

DNA Replication• DNA polymerases

– Catalyze synthesis of new DNA by adding nucleotides to preexisting chain

– Prokaryotes• DNA polymerase III & DNA polymerase I

– Eukaryotes• ~ 11 DNA polymerases identified

11

DNA Replication• Nucleoside triphosphate

– Sugar, base + 3 phosphate groups– Removal of 2 phosphates catalyzed by DNA polymerase III

• Nucleotides can only be added at 3’ end• Elongation in 5’ to 3’ direction

Fig. 16.14

12

Leading and Lagging Strands

• Leading strand– The new complementary DNA strand synthesized

continuously along the template strand toward the replication fork

– DNA polymerase III and sliding clamp

Fig. 16.15

13

Leading and Lagging Strands

Lagging strand• A discontinuously synthesized

DNA strand that elongated by means of Okazaki fragments– A short segment of DNA

synthesized away from the replication fork

• 100-200 nucleotides (eukaryotes)

• Requires multiple primers

Fig. 16.16

14

Leading and Lagging Strands

• DNA polymerase I– Replaces RNA nucleotides

of primer with DNA nucleotides

• DNA ligase– Joins the Okazaki

fragments

Fig. 16.16

15

DNA Repair

• Errors in completed DNA molecule– 1 in 10 billion nucleotides

• Initial pairing errors in DNA replication– 1 in 100,000 nucleotides

• Corrections during replication– DNA polymerase proofreads

• If error in match, nucleotide removed and replaced

– Mismatch repair• Repair by other enzymes if DNA

polymerase missed the error

16

DNA Repair

• Corrections after replication– ~100 repair enzymes identified in in

E. coli– ~130 repair enzymes identified in

humans• Nucleotide excision repair

– E.g., repair of thymine dimers• Covalent linking of adjacent thymine

bases• Causes DNA to buckle• Caused by UV radiation

– Nuclease cuts damaged DNA at two points

– DNA polymerase adds nucleotides– DNA ligase joins nucleotides

Fig. 16.18

17

DNA Repair

Replicating ends of linear DNA molecules

• Nucleotides can only be added at 3’ end of existing strand

• No way to replace the primer on the 5’ end

• Linear DNA molecules grow shorter with each replication– In somatic cells

Fig. 16.19

18

DNA Repair

• Telomeres– Repeating sequence of nucleotides at ends of

linear chromosomes• TTAGGG in humans• Repeated 100 to 1000 times

– Do not contain genes– Chromosomes continue to shorten– Cell eventually dies

19

DNA Repair

Preserving DNA ends in meiosis

• Telomerase– Catalyzes lengthening

of telomeres in germ cells

– Preserves length of chromosomes in gametes

– Not active in most somatic cells

20

DNA Repair

• Telomerase and cancer– Chromosomes of somatic cells gradually

shorten• Telomere loss signals cells to enter non-dividing

stage

– If telomerase activated in somatic cells, cell may continue to divide

• May become cancerous

21