DNA Replication

But first a little review!

DNA is made up of subunits called NUCLEOTIDES or

each nucleotide is made up of 3 basic parts: * 5-carbon sugar: deoxyribose * nitrogenous base: A, G, C, or T * 1 phosphate group

a) DNA is a helix with a width of 2 nm

b) purine & pyrimidine bases are stacked 0.34 nm apart

c) the helix makes 1 full turn every 3.4 nm along its length

d) there are 10 layers of bases (or rungs) in each turn of the helix

Watson and Crick Calculations based on Franklin’s work

to be consistent with a 2 nm width, a purine on one strand must pair (by H-bonding) with a pyrimidine on the other strand

base structure dictates which pairs of bases can form hydrogen bonds

LE 16-UN298

Purine + purine: too wide

Pyrimidine + pyrimidine: too narrow

Purine + pyrimidine: widthconsistent with X-ray data

LE 16-8

Adenine (A) Thymine (T)

Guanine (G) Cytosine (C)

Sugar

Sugar

Sugar

Sugar

LE 16-9_3

The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.

The first step in replication is separation of the two DNA strands.

Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.

DNA Replication

“The Beauty of the model was that the structure of DNA suggested the basic mechanism of its replication”

Watson and Crick proposed that during DNA replication: (2nd paper) 1) the 2 DNA strands separate(hydrogen bonds break; unwind; unzip DNA strand)

2) each strand is a template for assembling a complementary strand

Watson and Crick proposed that during DNA replication: (2nd paper)

3) nucleotides line up singly along the template strand (A-T, G-C) (new hydrogen bonds form)

4) enzymes link the nucleotides together at their sugar-phosphate groups (phosphodiester bonds)

Watson and Crick’s proposed model is a SEMICONSERVATIVE MODEL (each of the 2 daughter molecules will have 1 old or CONSERVED strand from the parent molecule and 1 newly created strand)

*RECALL: DNA strands run in

opposite directions

(antiparallel)

1 strand is made continuously (LEADING STRAND)

and…

the second strand is made discontinuously (in fragments) (LAGGING STRAND)

the LAGGING STRAND is produced as a series of short fragments (“Okazaki” fragments) which are synthesized in the 5’ to 3’ direction.

5’ to 3’ DNA Strand

Structure of Nucleic AcidsStructure of Nucleic Acids • Nucleic acids are macromolecules built of

monomers called NUCLEOTIDES

Each nucleotide consists of:1. 5-carbon sugar:

-deoxyribose in DNA

 2.   Phosphate group (attached to #5 carbon on sugar) 

3.   Nitrogenous base– purines (double ring; A & G)

  

– pyrimidines (single ring; C, T & U)

3 Differences Between DNA and RNA nucleotides:

1) sugar in RNA is ribose

2) 2’ Carbon on Ribose has (OH group) and on Deoxyribose there is just a Hydrogen

3) DNA has the base thymine (T), RNA has the base uracil

(U)

• nucleotides are joined together by phosphodiester linkages (between phosphate of one nucleotide and the sugar of the next)

• this results in a backbone with a repeating pattern of: sugar-phosphate-sugar-phosphate...

Dehydration Synthesis

To Build the new DNA strands you will need some Deoxyribonucleotide Triphosphates (dNTP’s)  

each dNTP is made up of 3 basic parts: *5-carbon sugar: deoxyribose *nitrogenous base: A, G, C, or T *3 phosphate groups

The nucleotide subunits of DNA comprise the four letters of the genetic code

dCTP

dTTP

C

T

dATP

dGTP

A G

dATP = Deoxyadenosine triphosphatedGTP= Deoxyguanosine triphosphatedTTP= Deoxythymidine triphosphatedCTP = Deoxycytidine triphosphate

What are these for anyway???????????

These Nucleotide Triphosphates drive the DNA replication process. It provides energy for DNA synthesis:

-nucleotide triphosphate loses 2 phosphates

-Loss of these phosphates drives the synthesis of DNA; it provides the energy to form new covalent linkages

between nucleotides (also a dehydration synthesis reaction!—why?)

(lets look at link)

LE 16-13

New strand

5 end

Phosphate Base

Sugar

Template strand

3 end 5 end 3 end

5 end

3 end

5 end

3 end

Nucleosidetriphosphate

DNA polymerase

Pyrophosphate

But you also need lots of enzymes!..

DNA Replication:The Process

Lots of Enzymes involved! Major players:

1)Helicase2)Single-strand binding

proteins3)DNA polymerase III4)DNA polymerase I5)DNA Ligase6)Primase7)Nuclease

DNA replication begins at special sites called ORIGINS OF REPLICATION.

-DNA double helix opens at the origin & replication “forks” spread in both directions away from the central initiation site creating a REPLICATION BUBBLE.

  -100’s to 1000’s of replication origins form in eukaryotic chromosomes, which eventually fuse forming 2 continuous DNA molecules

Synthesizing a new DNA strand:

2 types of proteins involved with separation of parental DNA strands:

*HELICASES: unwinding/unzip the parental DNA double helix to expose templates

*Single-strand binding proteins: keep separated strands apart & stabilize unwound DNA until new strands can be made

Before new DNA strands can form, there must be small pre-existing PRIMERS to start the addition of new nucleotides

 -a primer is a short RNA segment that

is complementary to a DNA segment

-primers are put together (added) by the primase enzyme

-only 1 primer is necessary for replication of the leading strand, but many primers are necessary to replicate the lagging strand

*an RNA primer must initiate the synthesis of each Okazaki fragment

*

DNA polymerases catalyze the synthesis of the new DNA

strand:-new nucleotides (2 phosphates were removed) align themselves along templates of old DNA strands according to base-pairing rules (A-T, G-C)

-DNA polymerase III links the nucleotides to growing strand.

-new nucleotides are added only to the 3’ end of the primer

(-OH end)

DNA polymerase III can only add

on the 3' end!

DNA polymerases catalyze the synthesis of the new DNA

strand:-The new strands grow in the 5’ to 3’ direction

DNA polymerase III will continue to add nucleotides to the 3’ end until it hits another primer (lagging strand only). Leading strand is just keeps going!

DNA polymerase III falls off when it hits another primer segment.

*RECALL: polymerase III can elongate

strands only in the 5’ to 3’ direction

LE 16-15_2

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

LE 16-15_3

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

Okazakifragment

3

5

5

3

After reaching thenext RNA primer (not

shown), DNA pol IIIfalls off.

*DNA polymerase I removes the RNA primers and replaces them with DNA bases

LE 16-15_2

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

LE 16-15_3

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

Okazakifragment

3

5

5

3

After reaching thenext RNA primer (not

shown), DNA pol IIIfalls off.

LE 16-15_4

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

Okazakifragment

3

5

5

3

After reaching thenext RNA primer (not

shown), DNA pol IIIfalls off.

33

5

5

After the second fragment isprimed, DNA pol III adds DNAnucleotides until it reaches thefirst primer and falls off.

LE 16-15_5

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

Okazakifragment

3

5

5

3

After reaching thenext RNA primer (not

shown), DNA pol IIIfalls off.

33

5

5

After the second fragment isprimed, DNA pol III adds DNAnucleotides until it reaches thefirst primer and falls off.

33

5

5

DNA pol I replaces the RNA with DNA,adding to the 3 endof fragment 2.

the enzyme DNA ligase then links the segments together by phosphodiester bonds therefore completing the strand.

LE 16-15_6

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

Okazakifragment

3

5

5

3

After reaching thenext RNA primer (not

shown), DNA pol IIIfalls off.

33

5

5

After the second fragment isprimed, DNA pol III adds DNAnucleotides until it reaches thefirst primer and falls off.

33

5

5

DNA pol I replaces the RNA with DNA,adding to the 3 endof fragment 2.

33

5

5

DNA ligase forms abond between the newestDNA and the adjacent DNAof fragment 1.

The lagging strand in the regionis now complete.

LE 16-16

5

3Parental DNA

3

5

Overall direction of replication

DNA pol III

Replication fork

Leadingstrand

DNA ligase

Primase

OVERVIEW

PrimerDNA pol III

DNA pol I

Laggingstrand

Laggingstrand

Leadingstrand

Leadingstrand

LaggingstrandOrigin of replication

Enzymes proofread DNA during its replication and repair damage to

existing DNA• Enzyme- Nuclease –cuts out

damaged or mismatched nucleotides

• Pol III adds correct nucleotide and Ligase makes final bond

END

What enzyme adds a DNA nucleotide to an existing nucleotide in DNA elongation?

A. DNA polymerase III

B. DNA polymerase I

C. Primase

D. Helicase

E. DNA Ligase

What kind of primer does primase make?

A. DNA

B. RNA

C. Single-stranded binding protein

D. Ligated

What does helicase do?

A. Form phosphodiester bonds

B. Hold DNA strands apart

C. Replace RNA nucleotides with DNA nucleotides

D. Unwind & separate DNA strands

Why must DNA elongate in the 5’ to 3’ direction?A. DNA ligase can only form covalent bonds at these Carbon

locationsB. DNA Polymerase III can only add a nucleotide to the 3’ end

of another nucleotideC. DNA Polymerase III can only add a nucleotide to the 5’ end

of another nucleotideD. DNA Polymerase I can only add a nucleotide to the 5’ end of

another nucleotideE. DNA Polymerase I can only add a nucleotide to the 3’ end of

another nucleotide

The Leading strand…

A. is formed in the 5’ to 3’ directionB. is elongating into a replication forkC. is elongating away from a replication forkD. is composed of Okazaki fragmentsE. Both A and BF. Both C and D

Enzyme Function1.

2.

3.

4.

5.

6.

7.

LE 16-15_2

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

LE 16-15_3

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

Okazakifragment

3

5

5

3

After reaching thenext RNA primer (not

shown), DNA pol IIIfalls off.

LE 16-15_4

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

Okazakifragment

3

5

5

3

After reaching thenext RNA primer (not

shown), DNA pol IIIfalls off.

33

5

5

After the second fragment isprimed, DNA pol III adds DNAnucleotides until it reaches thefirst primer and falls off.

LE 16-15_5

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

Okazakifragment

3

5

5

3

After reaching thenext RNA primer (not

shown), DNA pol IIIfalls off.

33

5

5

After the second fragment isprimed, DNA pol III adds DNAnucleotides until it reaches thefirst primer and falls off.

33

5

5

DNA pol I replaces the RNA with DNA,adding to the 3 endof fragment 2.

LE 16-15_6

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

Okazakifragment

3

5

5

3

After reaching thenext RNA primer (not

shown), DNA pol IIIfalls off.

33

5

5

After the second fragment isprimed, DNA pol III adds DNAnucleotides until it reaches thefirst primer and falls off.

33

5

5

DNA pol I replaces the RNA with DNA,adding to the 3 endof fragment 2.

33

5

5

DNA ligase forms abond between the newestDNA and the adjacent DNAof fragment 1.

The lagging strand in the regionis now complete.

LE 16-16

5

3Parental DNA

3

5

Overall direction of replication

DNA pol III

Replication fork

Leadingstrand

DNA ligase

Primase

OVERVIEW

PrimerDNA pol III

DNA pol I

Laggingstrand

Laggingstrand

Leadingstrand

Leadingstrand

LaggingstrandOrigin of replication

LE 16-15_1

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

MISMATCH REPAIR: corrects mistakes when DNA is copied

*one form of colon cancer is due to a defect in one of the proteins involved in this type of DNA repair

EXCISION REPAIR: corrects accidental changes that occur in existing DNA

-an enzyme (nuclease) cuts out damaged segment of DNA

-enzymes DNA polymerase III and ligase fill in the resulting gaps

*xeroderma pigmentosum (XP) is caused by an inherited defect

in an excision-repair enzyme

Sugar + Base. A nucleoside consists of a nitrogenous base covalently attached to a sugar (ribose or deoxyribose) but without the phosphate group.

Nucleoside Nucleotide

Sugar + Base + Phosphate. A nucleotide consists of a nitrogenous base, a sugar (ribose or deoxyribose) and one to three phosphate groups.

• Each nucleotide consists of:1. Pentose (5-carbon sugar)

-deoxyribose in DNA-ribose in RNA

 2.   Phosphate group (attached to #5 carbon on sugar) 

3.   Nitrogenous base– purines (double ring; A & G)

  

– pyrimidines (single ring; C, T & U)

the LAGGING STRAND is produced as a series of short fragments (“Okazaki” fragments) which are synthesized in the 5’ to 3’ direction and then linked together by the enzyme DNA ligase.