AP Biology

Mr. Anderson: Replication (https://www.youtube.com/watch?v=FBmO_rmXxIw)

AP Biology

Copying DNA – S PhaseReplication occurs during the S-Phase of the cell

cycle. Each DNA molecule is used to

synthesize identical daughter strands of DNA.

The cell must replicate it's DNA so that there will

be enough genetic material for new cells

formed through mitosis, to each have a correct

and identical set of DNA.

Replication occurs in the nucleus of Eukaryotic

cells and the cytoplasm of Prokaryotic

AP Biology

Basic Replication animation

http://www.bioteach.ubc.ca/TeachingResources/MolecularBiology/DNAReplication.swf

Steps involved in Replication:

*remember - genetic information is stored in

the order of the bases in the rungs of DNA.

So in order to replicate the information the

double stranded DNA molecule must open

so the order of the bases can be replicated.

AP Biology

http://student.ccbcmd.edu/~gkaiser/biotutorials/dna/fg12.html

Link - Bacterial replication

Site where it starts = ORIGIN of REPLICATION -

DNA strands are separated, opening up a

replication 'bubble'.

Place where nucleotides add = REPLICATION

FORK .

Note that there is a replication fork at each end of

the bubble.

Prokaryote- single starting spot

Eukaryotes-multiple sites

AP Biology

The DNA Replication Complex

• The proteins that participate in DNA

replication form a large complex, a “DNA

replication machine”

• The DNA replication machine may be

stationary during the replication process

• Recent studies support a model in which

DNA polymerase molecules “reel in”

parental DNA and “extrude” newly made

daughter DNA molecules

© 2011 Pearson Education, Inc.

AP Biology

Figure 16.18

Parental DNA

DNA pol III

Leading strand

Connectingprotein

Helicase

Lagging strandDNA pol III

Laggingstrand

template

5

5

5

5

5

5

3 3

33

3

3

AP Biology

The DNA Replication Complex includes:

Enzymes called DNA polymerases catalyze the

elongation of new DNA at a replication fork

Most DNA polymerases require a primer and a

DNA template strand

The rate of elongation is about 500 nucleotides

per second in bacteria and 50 per second in

human cells

© 2011 Pearson Education, Inc.

AP Biology

DNA POLYMERASE III

• reads code strand in 3’ → 5’ direction

• builds a new strand in 5’→3’ direction

• adds on to 3’ end of sugar in previous

nucleotide

AP Biology

Energy of Replication Splitting phosphates from nucleotide triphosphate subunits

provides energy for the replications reaction

The nucleotides arrive as nucleoside triphosphates

DNA bases with P–P–P P-P-P = energy for bonding

DNA bases arrive with their own energy source for bonding

bonded by enzyme: DNA polymerase III

ATP GTP TTP CTP

AP Biology

energy

ATPGTPTTPCTP

Energy of Replication

Where does energy for bonding usually come from?

ADPAMPGMPTMPCMP

modified nucleotide

energy

We comewith our own

energy!

And weleave behind anucleotide!

Youremember

ATP!Are there other ways

to get energyout of it?

Are thereother energynucleotides?

You bet!

AP Biology

DNA

Polymerase III

Adding

Complementary

Bases

Where’s theENERGY

for the bondingcome from?

AP Biology

can only add

nucleotides to

3 end of an

existing

DNA/RNA Chain,

therefore, a new

DNA strand can

elongate only

grows 53

DNA

Polymerase III

DNA

Polymerase III

DNA

Polymerase III

DNA

Polymerase III

energy

energy

energy

DNA POLYMERASE

CAN’T START A CHAIN

by itself;

energy

3

3

5

5

AP Biology

Replication Enzymes HELICASE- untwists double helix to open strands at

replication forks

TOPOISOMERASE- relieves strain caused by untwisting,

breaking and rejoining DNA strands.

SINGLE-STRAND BINDING PROTEINS-

stabilize unpaired strands to hold them open

single-stranded binding proteinsreplication fork

helicase

Link: DNA

REPLICATION FORK

AP Biology

Replication Enzymes PRIMASE-starts segment by adding a primer made up of 5 -

10 RNA nucleotides, and the 3' end serves as a starting

point for the new DNA strand.

*Evolutionary significance - Since a

primer is made from RNA and can be

used as a template for DNA, it suggests

that RNA existed before DNA.

AP Biology

Replication Enzymes

DNA POLYMERASE I – removes RNA

primers and replaces them with DNA

bases by adding to the 3’ end of the

previous fragment

LIGASE-joins Okazaki fragments together

to make a continuous copied strand

AP Biology

energy

35

5

5

3

need “primer” bases to add on to

energy

energy

energy

3

no energy

to bond

energy

energy

energy

ligase

3 5

AP Biology

Limits of DNA polymerase III

can only build onto 3 end of

an existing DNA strand

Leading & Lagging strands

5

5

5

5

3

3

3

5

35

3 3

Leading strand

Lagging strandligase

Okazaki

Leading strand

continuous synthesis

Lagging strand

Okazaki fragments

joined by ligase

“spot welder” enzyme

DNA polymerase III

3

5

growing replication fork

AP Biology

LEADING STRAND

(Original DNA strand runs 3’→ 5’) copies

toward the replication fork

PRIMASE adds RNA primer to start

chain

DNA POLYMERASE III adds nucleotides

in 5’ → 3’ direction (referring to new

molecule)

AP Biology

DNA polymerase III

Replication fork / Replication bubble

5

35

3

leading strand

lagging strand

leading strand

lagging strandleading strand

5

3

3

5

5

3

5

3

5

3 5

3

growing replication fork

growing replication fork

5

5

5

5

5

3

3

5

5lagging strand

5 3

AP Biology

LAGGING STRAND

(Original DNA strand runs 5’→ 3’) copies

away from replication fork

PRIMASE adds RNA primers at various

spots as fork opens

DNA POLYMERASE III adds nucleotides

in 5’ → 3’ (referring to new molecule)

direction in short segments= OKAZAKI

FRAGMENTS

AP Biology

DNA polymerase III

RNA primer

built by primase

serves as starter sequence for DNA polymerase III

Limits of DNA polymerase III

can only build onto 3 end of

an existing DNA strand

Starting DNA synthesis: RNA primers

5

5

5

3

3

3

5

35

3 5 3

growing replication fork

primase

RNA

AP Biology

DNA polymerase I

removes sections of RNA

primer and replaces with

DNA nucleotides

But DNA polymerase I still

can only build onto 3 end of

an existing DNA strand

Replacing RNA primers with DNA

5

5

5

5

3

3

3

3

growing replication fork

DNA polymerase I

RNA

ligase

Link: HOW NUCLEOTIDES

ARE ADDED

AP Biology

Loss of bases at 5 ends

in every replication

chromosomes get shorter with each replication

limit to number of cell divisions?

DNA polymerase III

All DNA polymerases can

only add to 3 end of an

existing DNA strand

Chromosome erosion

5

5

5

5

3

3

3

3

growing replication fork

DNA polymerase I

RNA

Houston, we have a problem!

TELOMERES & TELOMERASE

Image from: AP BIOLOGY by Campbell and Reese 7th edition

Primer removed but

can’t be replaced with

DNA because no

3’ end available for

DNA POLYMERASE

IMPORTANT:

Because DNA

polymerase can’t fill

in last section when

primer is removed

from lagging strand,

the code shortens

with each

replication (usually only a

problem for Eukaryotes since

prokaryotes have circular

chromosomes)

Eukaryotic chromosomal DNA molecules have

special nucleotide sequences at their ends

called telomeres.

TELOMERE sequences at ends of

chromosomes to help postpone the erosion of essential information in code / genes with

each replication.

• it has been proposed that the shortening of

telomeres may play a role in a aging and

cancer

TELOMERASE = enzyme that lengthens

telomeres • found in eukaryotic germ cells that divide

frequently to produce gametes

Link 2: TELOMERES-http://stemcells.nih.gov/info/scireport/appendixC.asp

Link 3: ANIMATIONLink 1:

AP Biology

PROOFREADING & REPAIR

Mistakes in final DNA: 1 in 10 billion

Mistakes in initial base pairing during

replication 1 in 100,000

DNA POLYMERASE proofreads each base as it’s

added & fixes errors

Errors can come from “proofreading

mistakes” that are not caught OR

environmental damage

(Ex: X-rays, UV light, chemical mutagens/

carcinogens)

NUCLEOTIDE EXCISION REPAIR Cells continually monitor

DNA and make repairs

1. NUCLEASES- DNA

cutting enzymes

remove errors

2. DNA POLYMERASE fills

in gap using

complimentary strand

3. LIGASE seals ends

AP Biology

Ex: THYMINE DIMERS

= joins THYMINES in same

strand

• damage caused by UV

light

• can be repaired

AP Biology

Evolutionary Significance of

Altered DNA Nucleotides

Error rate after proofreading repair is

low but not zero

Sequence changes may become

permanent and can be passed on to the

next generation

These changes (mutations) are the

source of the genetic variation upon

which natural selection operates

AP Biology

Replication fork

3’

5’

3’

5’

5’

3’

3’ 5’

helicase

direction of replication

SSB = single-stranded binding proteins

primase

DNA polymerase III

DNA polymerase III

DNA polymerase I

ligase

Okazaki fragments

leading strand

lagging strand

SSB

AP Biology

DNA polymerases

DNA polymerase III

1000 bases/second!

main DNA builder

DNA polymerase I

20 bases/second editing, repair & primer removal

DNA polymerase III enzyme

Arthur Kornberg1959

Thomas Kornberg

http://bio.usuhs.mil/biochem4.html

Replication Summary.

Go trough the following animation to review

the entire process of replication:

Link:

http://sites.fas.harvard.edu/~biotext/animatio

ns/replication1.swf

Be sure to complete the four parts –

a) Replication fork

b) Fork with proteins

c) Concerted replication

d) Trombone model