L8 Dna Replication

Front 1

What is an overview of DNA replication?

Back 1

DNA unwinds

Daughter strands ofDNA produced fromparent strand atreplication fork

Semi conservativereplication (eachnew DNA moleculecontains one parentand one daughterstrand)

Front 2

Is the DNA formed identical to the parent DNA?

Back 2

An identical copy of the parentDNA is synthesised

Front 3

How are new nucleotides added to the free 3'OH group of the growing polynucleotide strand?

Back 3

New nucleotides are added to the free 3’OH group of the growing polynucleotidestrand through formation of newphosphodiester bonds by an enzyme,DNA polymerase

Front 4

Where is energy for this process derived from?

Back 4

Energy required forthis processderived fromhydrolysis of highenergy bonds ofnucleotidetriphosphates

Front 5

What are the features of DNA replication?

Back 5

DNA polymerase requires asingle stranded template

DNA must be unwound

In eukaryotic cells, DNA mustbe removed from histoneproteins

Front 6

Where is the initiation of replication?

Back 6

Initiation of replication by proteinsinteracting with DNA sequences atthe “origin of replication”

Front 7

How are two "replication forks" formed?

Back 7

Helicase enzymesare able toseparate the twostrands of DNA

Two “replicationforks” are formed

Front 8

How does replication proceed?

Back 8

Replication proceeds as thereplication forks move, unwindingdouble stranded DNA

Front 9

What uncoils the supercoiled DNA ahead of replication?

Back 9

Topoisomerase enzymesuncoil the supercoiled DNAstrands ahead of replication•Type I topoisomeraserelaxes supercoil DNA•Type II topoisomerase(gyrase) cuts andreseals the DNA strandsto relieve supercoiling

Front 10

In what direction does DNA polymerase make new DNA?

Back 10

DNA polymerase IIImakes new DNA in5’ ’ → 3’ direction

Front 11

What is the polarity problem?

Back 11

DNA polymerasescan only make DNA chains in the 5’-> 3’ direction.

Since the strands of DNA are anti-parallel, in order for both strands of template DNA to be replicated at the sametime, one strand has to be copied in the 5’->3’ direction and the other in the 3’->5’ direction.

So, how do both strands get replicated using DNA polymerases that can make chains in the 5’-> 3’ direction only?

Front 12

What is the solution to the polarity problem?

Back 12

The leading strand issynthesisedcontinuously butthe lagging strandis synthesised asa series of shortfragments

1. RNA primeraddition

2. DNA polymerasesynthesisesfragments in 5’ →3’ direction

Front 13

Why is the lagging strand looped?

Back 13

The lagging strand template islooped to allow lagging strandsynthesis

Front 14

How does the looped strand allow lagging strand replication?

Back 14

DNA polymerase IIImakes new DNA onboth strandssimultaneously andmoves in samedirection as replicationfork.

DNA on laggingstrand must be loopedout to allow this

Front 15

What does the Single strand binding protein?

Back 15

Stabilises single stranded DNA in separatedstrands

Front 16

What does helicase do?

Back 16

Unwinds DNA strands at the replication fork

Front 17

What does the B protein (sliding clamp) do?

Back 17

Attaches DNA pol III firmly to the DNA

Front 18

What does DNA polymerase III do at the leading strand?

Back 18

synthesises new DNA strand

Front 19

What does primase do at the lagging strand?

Back 19

Makes RNA primer so that DNA synthesis canbegin

Front 20

What does DNA polymerase III do at the lagging strand?

Back 20

Synthesises new DNA strand UNTIL itreaches the RNA primer of a precedingOkazaki fragment

Front 21

What does DNA polymerase I do at the lagging strand?

Back 21

Removes nucleotides of RNA primer andadds deoxynucleotides to fill the gap withDNA

Front 22

What does DNA ligase do?

Back 22

Joins discontinous DNA fragments bymaking a phosphodiester bond

Front 23

Can the new DNA be an imperfect copy?

Back 23

Newly synthesised DNA must bea perfect copy

Front 24

How can mutations arise?

Back 24

 Can be created by incorporation of incorrectnucleotides into new DNA chain

 1 base in 7000 is wrong

Front 25

What detects mutations in the new DNA?

Back 25

DNA polymerases I and III haveproofreading function

Front 26

How is the Mutation corrected?

Back 26

 Detect incorrect base pair (differentshape)

 3’ – 5’ exonuclease activity removesincorrect base

 Polymerase inserts correct base instead

Front 27

What happens when a DNA mutation is found in E.coli?

Back 27

 Distortions in double helix detected by acomplex of proteins

 The complex can distinguish between newDNA and template DNA

 Another group of proteins remove a sectionof newly formed DNA (1000s of bases)

 DNA pol III re-synthesises the new DNAstrand

Front 28

How many errors are there in DNA despite the action of DNA Pol

Back 28

With DNA Pol proofreading and the DNA repairmechanisms, overall the final error rate is 1 in 1010

only 1 out of every 10 000 000 000 bases are wrong

Front 29

What can be the consequences of single base mutations?

Back 29

 Consequences of single base mutations can bevery serious

 Mutated gene = abnormal protein = loss ofimportant function OR gain of toxic function

 e.g. many cancers caused by small mutations inproteins that control cell growth

Front 30

What happens when DNA is damaged?

Back 30

DNA can be damaged, and isrepaired by DNA repair complexes

Front 31

What happens when DNA is damaged by UV light?

Back 31

Ultraviolet light

Thymine dimer formation

Uncorrected, can lead to point mutations that cause skincancer

Front 32

What happens when DNA is damaged by carcinogenic chemicals?

Back 32

Carcinogenic chemicals e.g. in tobacco smoke Cause various chemical modifications to bases Uncorrected can lead to point mutations that cause lungcancer

Front 33

What does Ionising radiation cause to happen?

Back 33

strand breaks

Front 34

What does Nitrous acid cause to happen?

Back 34

cytosine to uracil, CG → TA

Front 35

What do Alkylating agents cause to happen?

Back 35

guanine modification, GC → AT

Front 36

What can Free radicals cause to happen?

Back 36

strand breaks and basemodification

Front 37

What do Bulky chemicals do?

Back 37

distort DNA helix

Front 38

What can happen in spontaneous mutation?

Back 38

cytosine to uracil, CG → T;

loss of purines

Front 39

What is Xeroderma pigmentosum?

Back 39

 Moon children

 Genetic disorder,lacking DNA repairmechanism

 Even modest exposureto sunlight causes skintumours

Front 40

How does DNA replication compare in Eunkaryotes and prokaryotes?

Back 40

 Processes very similar but more enzymes, named differently

 Roles of different polymerases still under investigation (so you need toknow that different polymerases exist and recognise the nomenclature,but do not need to memorise their suggested roles)

Front 41

What are the suggested roles of the polymerases?

Back 41

 DNA polymerase a

 Assembles primers and initiates synthesis of leading strand andOkazaki Fragments of lagging strand

 Lacks proofreading activity

 DNA polymerase B

 Involved in DNA repair

 DNA polymerase g

 Synthesises mitochondrial DNA

 DNA polymerase d

 Main synthesis of lagging strand

 Has proofreading activity

 DNA polymerase e

 Main synthesis of leading strand

Front 42

What are the Differences in eukaryotic andprokaryotic replication and DNArepair?

Back 42

Eukaryotic cells have mitochondria

Mitochondria have their own single circularchromosome which is similar to a bacterialchromosome

Mitochondrial DNA needs to be replicatedindependently from the nuclear chromosomesas eukaryotic cells divide.

In eukaryote cells, DNA replication ispart of the “cell cycle”

In eukaryotic cells, there is formationof numerous replicons inchromosomal DNA

Front 43

What is the eukaryotic cell cycle?

Back 43

 Interphase

 G1 phase: synthesis ofprotein componentsnecessary to completethe cell cycle includinghistones, enzymes forreplication etc.

 S phase: DNAsynthesis commences.Replicons areobserved.