Lecture 4

Question 1

DNA strands serve as…

Answer 1

• template
• arrangement and nature of nitrogenous bases allow DNA strands to serve as templates
• complementarity of DNA strands allows each strand to serve as template for synthesis of the other

Question 2

Semiconservative model of DNA replication

Answer 2

each replicated DNA molecule consists of one old and one new strand

Question 3

Conservative model of DNA replication

Answer 3

two newly synthesized strands come together and the original helix is conserved

Question 4

Dispersive model of DNA replication

Answer 4

parental strands are dispersed into 2 new double helices

Question 5

Meselson and Stahl experiment

Answer 5

• 15N labeled E.coli grown in medium containing 14N
• each new DNA molecule consists of one old and one newly synthesized stand
• provided strong evidence that DNA is semiconservative in prokaryotes

Question 6

Taylor-Woods-Hughes experiments

Answer 6

• Vicia faba (broad bean) was used to demonstrate DNA replication is semiconservative in eukaryotes
• monitored process of replication with labeled 3H-thymidine and performed autoradiography

Question 7

Where does DNA replication begin?

Answer 7

At the ORI - origin of replication

Question 8

What is made at the ORI?

Answer 8

the replication fork which is the product of the unwound helix

Question 9

How many replication forks are on a strand?

Answer 9

2 because replication is bidirectional

Question 10

Replicon

Answer 10

length of DNA replicated

Question 11

How many ORI does bacteria have?

Answer 11

1
- singular circular DNA
- DNA synthesis originates at OriC
- E. Coli replicon consists of entire genome of 4.6 million base pairs

Question 12

Details about elongation by DNA polymerase I

Answer 12

• occurs in the 5’ to 3’ direction by adding one nucleotide at a time to 3’ end
• nucleotide added, two terminal phosphates cleaved off, providing newly exposed 3’ OH
• 3’ OH can participate in addition of another nucleotide as DNA synthesis proceeds

Question 13

DNA Pol: I, II, III

Answer 13

• can elongate existing DNA strand
• cannot initiate DNA synthesis without a RNA primer

Question 14

Exonuclease activity 3’-5’

Answer 14

DNA pol I, II, III all posses 3’-5’ exonuclease activity: proofread newly synthesized DNA, remove/replace incorrect nucleotides

Question 15

Exonuclease activity 5’-3’

Answer 15

• only DNA polymerase I removes RNA primer
• excises primer - fills in gaps left behind

Question 16

What are 7 keys issues that need to be resolved during DNA replication?

Answer 16

1) Unwinding of helix
2) Reduce increased coiling generated during unwinding
3) Synthesis of primer for replication
4) Discontinuous synthesis of second strand
5) Removal of RNA primers
6) Joining of gap filling DNA to adjacent strand
7) proofreading

Question 17

DnaA

Answer 17

• inhibitor protein encoded by gene dnaA
• Binds to ORI causing conformation change
• causes helix to open up and destabilize
• exposed ssDNA

Question 18

DNA helicase

Answer 18

• made of DnaB polypeptides
• Hexamer of subunits: assembles around exposed ssDNA
• Subsequently recruits holoenzyme to bind replication fork and initiate replication
• Helicases require energy supplied by hydrolysis of A T P—denatures hydrogen bonds and stabilizes double helix

Question 19

Single-stranded binding proteins (SSBPs)

Answer 19

– Stabilize the open conformation of helix
– Bind specifically to single strands of D N A

Question 20

DNA Gyrase

Answer 20

– Enzyme relieves coiled tension from unwinding of helix (DNA supercoiling)
– Member of larger enzyme group: DNA topoisomerases
– Makes single- or double-stranded cuts
– Driven by energy released during ATP hydrolysis

Question 21

Primase: RNA polymerase

Answer 21

– Recruited to replication form by helicase
– Synthesizes R N A primer
– Provides free 3’-O H required by DNA polymerase III for elongation

Question 22

DNA polymerase I

Answer 22

removes primers and replaces it with DNA

Question 23

RNA priming

Answer 23

• Universal phenomenon
• found in bacteria, viruses, and several eukaryotic organisms

Question 24

Double stranded DNA is….

Answer 24

antiparallel
5’-3’
3’-5-

Question 25

Which strand is made continuously during replication

Answer 25

leading strand

Question 26

Which strand is made in fragments during replication

Answer 26

lagging strand

Question 27

Okazaki fragments

Answer 27

lagging strand is made by Okazaki fragments each with an RNA primer

Question 28

DNA polymerase I

Answer 28

removes primer on lagging strand

Question 29

DNA ligase

Answer 29

- catalyzes formation of phosphodiester bonds - seals nicks and joins fragments

Question 30

Both strands are synthesized...

Answer 30

concurrently by a single replication fork

Question 31

During replication the lagging strand is synthesized

Answer 31

Looped - inverts physical but not biochemical direction

Question 32

DNA clamp

Answer 32

prevents core enzyme dissociation from template

Question 33

proof reading and error correction

Answer 33

- integral part of DNA replication - DNA polymerase is not always perfect - synthesis of non-complementary base pairs inserted occasionally - DNA polymerse exonuclease activity 3'to 5' allows for excise of nucleotides

Question 34

Which enzymes and proteins are essential for DNA synthesis

Answer 34

- DNA polymerase III core enzyme - SSBPS: single-stranded binding protein - DNA gyrase - DNA helicase - RNA primers

Question 35

Mutation

Answer 35

- interrupt or impair aspects of replication - ex) lethal mutations, ligase-deficient mutations, proofreading deficient mutations

Question 36

Conditional mutations

Answer 36

expressed under specific conditions

Question 37

temperature sensitive mutation

Answer 37

- Example of conditional mutation – May not be expressed at particular permissive temperature – Mutant cells grown at restrictive temperature and mutant phenotype expressed

Question 38

What features are shared between Eukaryotic and bacterial DNA

Answer 38

– Double-stranded D N A unwound at ORI – Replication forks formed – Bidirectional synthesis creates leading and lagging strands – Eukaryotic polymerases require four deoxyribonucleoside triphosphates, template, and prime

Question 39

Why is eukaryotic DNA replication more complex?

Answer 39

- More DNA than in prokaryotic cells - linear chromosomes - DNA complexed with nucleosomes

Question 40

Details about eukaryotic DNA replication

Answer 40

- eukaryotic chromosomes have multiple ORIs which facilitates rapid synthesis of large quantity of DNA

Question 41

What can control the timing of replication within eukaryotes

Answer 41

ORIs

Question 42

Prereplication complex - pre-RC

Answer 42

- assembles at replication of ORIs - early GI phase of cell cycle: - origin recognition complex (ORC) recognizes ORI's and tags ORI as site of initiation

Question 43

DNA polymerases involved in nuclear genome DNA replication

Answer 43

- pol alpha - pol beta - pol sigma involved in initiation and elongation

Question 44

Polymerase switching

Answer 44

occurs once the primers are put in place - pol alpha replaced by pol sigma and beta for elongation - pol beta synthesizes lagging strand - pol sigma synthesizes leading strand

Question 45

Telomeres

Answer 45

- inert chromosomal ends that protect intact eukaryotic chromosomes from improper fusion and degradation - long stretches of short repeating sequences preserve the integrity/stability of chromosome

Question 46

What stabilizes chromosome ends

Answer 46

telomere t-loops and a complex of six proteins binds and stabilizes chromosomes ends - this forms a shelterin complex

Question 47

Large chi-square means

Answer 47

data doesn't fit

Question 48

Small chi-square means

Answer 48

data does fit

Question 49

Telomerase

Answer 49

- eukaryotic enzyme - ribonucleoprotein: RNA synthesis of DNA complement - telomerase RNA component - telomerase reverse transcriptase - telomerase adds repeats of six-nucleotide sequences to 3' end to fill gaps

Question 50

When are there no longer and 3'-OH groups to elongate from?

Answer 50

once primer is removed on the lagging strand

Question 50

what happens to telomeres after division

Answer 50

they get shorter

Question 50

stem cells and malignant cells

Answer 50

maintain telomerase activity- immortalized

Question 50

in most eukaryotic cells telomerase...

Answer 50

is not active

Question 51

Genetic recombination and homologous recombination

Answer 51

genetic exchange at equivalent positions along 2 chromosomes with substantial sequence homology

Question 51

Telomerase activity and telomere length linked to...

Answer 51

aging , cancer, and other diseases

Question 52

Genetic recombination invovled:

Answer 52

- endonuclease nicking - strand displacement and pairing with complement - ligation - branch migration - duplex separation - generates characteristic Holliday structure

Question 53

Enzymes and proteins involved in homologous recombination

Answer 53

- RecA protein in E. Coli promotes exchange of reciprocal ssDNA molecules - bring about strand invasion and displacement