Replication

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NN H

R

OCH3

O

T

NN

R

HN H

O

C

R

N

N

N

N

HH N

A

G

R

N

N

N

H N

O

H N H

U

OHCH2 O OH

OH

OO P

O

OO P

O

OO P

O

OH

RiboseDeoxyRiboseDiDeoxyRibose

N

N

N

N

H N H

A

DeoxyAdenine (dA)

DeoxyAdenineTriphosphate (dATP)

DeoxyRibose

5’P

3’OH

3’OH

5’P3’OH 5’P

5’P 3’OH

3’ 3’ 3’

Just addingpolymerase

clamp loader, clamp, and polymerase

clamp and polymerase

primase,clamp loader, clamp, and polymerase

clamp loader, clamp, and polymerase

helicase

helicase,clamp loader, clamp, and polymerase

How much is 1010?

1 sheet/3000 characters * 1010 characters = 3,333,333 sheets1 ream/500 sheets * 3,333,333 sheets = 6666 reams1 box/10 reams * 6666 reams = 666 boxes

666 boxes of single-spaced typed sheets would fill the front of this room up to the ceiling with only a single spelling error.

Typical single space typewritten page 3000 characters/sheet

How fast is the fork going?E. coli can replicate in about 20 minutes under optimal conditions.E. coli genome contains 4.5*106 basepairs

4.5*106 basepairs/2 replication forks/1200 s =1875 basepairs/replication fork/s

By comparison….1 deck of cards/26 pairs1875 pairs * 1 deck of cards/26 pairs = 72 decks of cards

To move as fast as a replication fork you would have to be able to sort 72 decks of shuffled cards….pairing every club with a spade and every heart with a diamond…each second.

How many mistakes are made each time the cell replicates?

(1 genome/4.5*106 basepairs) * (1*1010 basepairs/1 error)=

H. sapiens genome 3.1*109 basepairs

E. coli genome 4.5*106 basepairs

(1 genome/ 3.1*109 basepairs) * (1*1010 basepairs/1 error)=

2222 genomes/error

3 genomes/error

What would happen in E. coli if mismatch repair did not occur?

What would happen in humans if mismatch repair did not occur?

Sorting it out And keeping things straight

2) Supercoiling helps to condense and pack the DNA, keeping it organized so that it can fit in the cell.

1) Partitioning functions help to keep things sorted/separated as replication occurs so that things aren’t too badly tangled when they finish.

Tools for examining / rearranging / changing the DNA

34

Nucleic Acid Hybridization

• DNA denaturation: Two DNA strands can be separated by heat without breaking phosphodiester bonds

• DNA renaturation = hybridization: Two single strands that are complementary or nearly complementary in sequence can come together to form a different double helix

• Single strands of DNA can also hybridize complementary sequences of RNA

Fig. 6.24

36

Restriction Enzymes

• Restriction enzymes cleave duplex DNA at particular nucleotide sequences

• The nucleotide sequence recognized for cleavage by a restriction enzyme is called the restriction site of the enzyme

• In virtually all cases, the restriction site of a restriction enzyme reads the same on both strands A DNA sequence with this type of symmetry is called a palindrome

Fig. 6.26

39

Southern Blot Analysis

• DNA fragments on a gel can often be visualized by staining with ethidium bromide, a dye which binds DNA

• Particular DNA fragments can be isolated by cutting out the small region of the gel that contains the fragment and removing the DNA from the gel.

• Specific DNA fragments are identified by hybridization with a probe = a radioactive fragment of DNA or RNA

• Southern blot analysis is used to detect very small amounts of DNA or to identify a particular DNA band by DNA-DNA or DNA-RNA hybridization

Fig. 6.27

Southern Blot Analysis

41

Polymerase Chain Reaction

• Polymerase Chain Reaction (PCR) makes possible the amplification of a particular DNA fragment

• Oligonucleotide primers that are complementary to the ends

of the target sequence are used in repeated round of denaturation, annealing, and DNA replication

• The number of copies of the target sequence doubles in each round of replication, eventually overwhelming any other sequences that may be present

42

Polymerase Chain Reaction

• Special DNA polymerase is used in PCR = Taq polymerase isolated from bacterial thermophiles which can withstand high temperature used in procedure

• PCR accomplishes the rapid production of large amounts of target DNA which can then be identified and analyzed

Polymerase chain reaction (PCR)

1) Needs only the smallest amount of DNA

2) Short DNA primers (that you can synthesize)

Heat

Polymerize

Cool

Allows you to amplify (generate a ton of) any gene or sequence that you need

45

DNA Sequence Analysis• DNA sequence analysis

determines the order of bases in DNA

• The dideoxy sequencing method employs DNA synthesis in the presence of small amounts of fluorescently labeled nucleotides that contain the sugar dideoxyribose instead of deoxyribose

Fig. 6.29

DNA Sequencing: Dideoxy Method

• Modified sugars cause chain termination because it lacks the 3’-OH group, which is essential for attachment of the next nucleotide in a growing DNA strand

• The products of DNA synthesis are then separated by electrophoresis. In principle, the sequence can be read directly from the gel

47

DNA Sequencing: Dideoxy Method

• Each band on the gel is one base longer than the previous band

• Each didyoxynucleotide is labeled by different fluorescent dye

• G, black; A, green; T, red; C, purple

• As each band comes off the bottom of the gel, the fluorescent dye that it contains is excited by laser light, and the color of the fluorescence is read automatically by a photocell and recorded in a computer

Fig. 6.31