Lecture 12: DNA Replication

Question 1

what way does the parental template strand run?

Answer 1

it runs 3’-5’

Question 2

Eukaryotic DNA replication

Answer 2

Multiple large linear
chromosomes
23 pairs in
humans

Multiple origins of
replication (ori)

Bidirectional

Question 3

what is needed to make a DNA copy?

Answer 3

Progressive addition of new nucleotides (A, C, T or G)

A starting point for nucleotide addition

Unwinding of the helical double-stranded DNA (to give two parental templates)

Release of tension generated by unwinding the DNA helix

Prevention of unwound double-stranded helical DNA, i.e. single-stranded DNA, from reforming and to protect it

Joining of ends of newly synthesised fragments together (lagging as well as leading strands)

Question 4

Leading Strand

Answer 4

Continuously synthesised in it’s 5’-3’

Question 5

Lagging strand

Answer 5

discontinuously synthesised in it’s 5’-3’ direction as Okazaki Fragments

Question 6

What is Primase?

Answer 6

Enzyme (a type of RNA polymerase) that makes an RNA primer = starting point for DNA polymerisation

Question 7

What is DNA Polymerase III?

Answer 7

Needs an OH group onto which the phosphate group of the incoming nucleotide can be attached

Only makes DNA
in the 5’ → 3’direction

Enzyme that synthesises a new DNA strand by adding nucleotides complementary to
the parental template strands

Cannot bind to single stranded DNA and start copying it

Question 8

What pulls the two dna strands apart?

Answer 8

the Helicase unzips the DNA

Question 9

what does the topoisomerase do?

Answer 9

goes ahead of the replication fork and cuts it to release the tension it will then glue them back together after the replication has proceeded

Question 10

what does the single stranded DNA binding protein do?

Answer 10

they keep the two split strands apart as the C-G and A-T want to go back together really bad.

their other function is that they prevent the single strands of dna from being degraded.

Question 11

what does primase do?

Answer 11

has an internal 3 prime hydroxyl group, they add a short stand of RNA nucleotides to the 3’ prime strand.

Question 12

what Dna pol III do?

Answer 12

it utalises the 3prime hydroxyl group from the primase and will add a dna strand all along the strand

Question 13

what does the primase do to the 3’ prime 5’ prime stand

Answer 13

it goes along and makes a bunch of strands for the DNA pol III to add dna strands inbetween

Question 14

what does DNA pol I do?

Answer 14

recognises the RNA DNA hybrid and goes along the strand and removes the RNA component

Question 15

What is ligase’s role in DNA replication?

Answer 15

used the 3 prime hydroxyl group and the 3 prime phosphate group to form a phosphodiester bond between the gaps

Question 16

What is needed to make a DNA copy revised:

Answer 16

DNA polymerase III
Primase Enzyme makes RNA Primer
Helicase
Topoisomerase nick and rejoins DNA strands
Single-stranded DNA binding protein
DNA polymerase I (RNase H activity removes RNA and DNA polymerase adds the DNA nucleotides)
DNA ligase

Question 17

When can DNA errors be repaired?

Answer 17

1. During replications (using an EXOnuclease)

2. After replication (Using an ENDonuclease)

Question 18

DNA replication shows high accuracy:

Answer 18

DNA pol III has a replication error rate of 1 in 10^8 base pairs replicated

Question 19

DNA pol III has a proofreading mechanism

Answer 19

chekcs the newly inserted nucleotide bases against the template

any incorrect bases are removed by a 3’ to 5’ EXOnuclease acitivity of DNA Pol III

Question 20

What can cause DNA damage or errors?

Answer 20

Incorrectly inserted bases are not corrected by DNA pol III

Radiation damage (e.g. UV)

Chemical modifications of bases (natural & chemical causes)

These types of incorrect or damaged nucleotide bases are removed by an ENDOnuclease

Question 21

What is the polymerase chain reaction?

Answer 21

In vitro method of making multiple DNA copies so that there is enough DNA material to work with

Only ‘targeted’ DNA region will be copied

Rapid exponential increase of DNA molecules

Method utilises cycles of heating and cooling

Question 22

What is an exonuclease and endonuclease and what is the difference between them?

Answer 22

Exonucleases are enzymes that catalyze the removal of nucleotides in either the 5-prime to 3-prime or the 3-prime to 5-prime direction from the ends of single-stranded and/or double-stranded DNA. Removal of nucleotides is achieved by cleavage of phosphodiester bonds via hydrolysis.

Endonuclease is an enzyme that breaks down a nucleotide chain into two or more shorter chains by cleaving the internal covalent bonds linking nucleotides — compare exonuclease.