Lec19

Question 1

Meselson and Stahl

Answer 1

tested the three hypothesizes for DNA replication by:

• Grew E. Coli in 15N media for many generations then they sampled the DNA
• Then transfer these cells to 14N Media
• Let the sample replicate for 1 generation then sample the DNA
• Let the sample replicate another generation then sample the DNA
• Find let the sample replicate a 3rd time and sample the DNA

DNA samples were analyzed by density gradient centrifugation (determined ratio between N14 and N15)

CONCLUDED DNA is SEMICONSERVATIVE

Question 2

Taylor, Woods, and Hughes

Answer 2

Different way to show that DNA was semiconservative
-used Autoradiography to examine chromosomes during (metaphase of mitosis) of replication

-3H Labeled Thymine only present for first cell division then pattern of radioactivity observed as cells continue to divide.
After the first cell division both chromatids show 3H, while after the second division only 1 chromatid contained the 3H labeling. WHICH SUPPORTED SEMICONSERVATIVE

Question 3

Theta replication

Answer 3

common in bacteria and other circular DNA molecules

Question 4

Rolling circle replication

Answer 4

with conjugation in the F factor and some viruses (ex. lambda)

Question 5

Linear Eukaryotic Replication

Answer 5

for eukaryotic chromosomes, which have multiple origins of replication along their length (eukaryotic replicons are 20,000-300,000 bp long)

Question 6

Where does all replication start at?

Answer 6

in the replication origin

Question 7

When does replication stop (proceeds to?)

Answer 7

until the entire REPLICON is replicated

Question 8

Replicon

Answer 8

unit that is replicated together

Question 9

What does Theta replication result in?

Answer 9

replication begins and then goes bidirectionally which results in 2 circular double stranded DNA molecules.

BIDIRECTIONAL REPLICATION

Question 10

Characteristics of rolling circle replication?

Answer 10

replication fork can continue around many times

-producing many strands that can then be used as templates such as double stranded DNA molecule

Question 11

Characteristics of linear eukaryotic replication?

Answer 11

• Multiple origins
• bidirectionally from each origin
• rate of 500-5000 nts/min
• Replicons typically 20,000 to 300,000 base pairs long
• unique procedure for replicating ends involving telomerase

Question 12

Telomerase

Answer 12

?

Question 13

DNA polymerase I in E. Coli

Answer 13

• discovered first
• requires template DNA, 4 deoxyribonucleoside, MG++ (used as cofactor)
• adds nucleotides 5’ to 3’ about 650 nts/min (thought to be slow)
• also has exonuclease function

Question 14

DNA polymerase II in E. Coli

Answer 14

• Adds 5’ to 3’

- Fills in gaps less than 100 nts (repairs DNA and is slower than polymerase I)

Question 15

DNA polymerase III in E. Coli

Answer 15

-Adds nucleotides 65,000 nt/min
-adds only in 5’ to 3’ direction
FASTEST

Question 16

What does a phosphodiester bond formation reaction require?

Answer 16

nucleoside triphosphates, not just nucleotides

Question 17

What catalyzes a phosphodiester bond formation reaction

Answer 17

catalyzed by the enzyme DNA polymerase

Question 18

Leading Strand

Answer 18

replication starts at the bottom and movies 3’-5’ of the template strand (continuous replication)

Question 19

lagging strand

Answer 19

replication starts at the top of the fork moving 3’-5’ of the template strand (discontinous replication)
-contains okazaki fragments

Question 20

Initiation: Replication process in E. Coli

Answer 20

-initiator protein (DnaA) binds to origon of replication causing local unwinding and a short stretch of single-stranded DNA

Question 21

Unwinding: replication process of E. Coli

Answer 21

• Helicase attaches to lagging template at replication fork and moves 5’-3’ breaking H-bonds as the replication fork moves along the DNA
• SSB binds to single stranded DNA stabilize it and prevent hairpin formation
• Gyrase rellieves supercoiling ahead of replication fork

Question 22

Topoisomerases

Answer 22

allows supercoiling to occur

-Gyrase is a type of topoisomerase

Question 23

Elongation Process

Answer 23

• DNA polymerase cannot start from scratch: They must have a 3’ OH that they can add nucleotides to.
• Primers are made by Primase, an RNA polymerase: ie. the primer is RNA
• DNA pol III adds nts primer
• The segment of RNA is eventually removed and replaced with DNA

Question 24

Primosome

Answer 24

helicase and primase??

Question 25

What is a nick?

Answer 25

nick is missing sugar phosphate bond

Question 26

Gap

Answer 26

missing a nucleotide

Question 27

purpose of DNA poly I

Answer 27

removes primer and fills in gap

Question 28

purpose of DNA ligase

Answer 28

seals nick

Question 29

ANALYZE CHART FOR REPLICATION FORK

Answer 29

!!!

Question 30

Many differences of Eukaryotic replication from prokaryotic replication.

Answer 30

- many origins of replication exist - origin must be licensed for replication to occur - linear chromosomes instead of 1 circular chromosome - Lots of different DNA polymerases molecules with various roles - Replication of telomeres to make sure the ends are properly replicated - Nucleosome assembly immediately follows replication

Question 31

What is the objective of Licensing of DNA replication

Answer 31

to make sure each piece of each chromosome is replicated once and only once per cell division in eukaryotes

Question 32

What doe the replication licensing factor do?

Answer 32

attaches to each origin of replication early in cell cycle

Question 33

Where will replication only start during licensing of DNA replication

Answer 33

will only START AT LICENSED ORIGINS | -as replication proceeds from origin, licensing factor is removed

Question 34

When is the licensing factor activated?

Answer 34

only active just after mitosis and before replication starts in cell cycle, so it will no be put back on an origin until early in the next cell cycle

Question 35

What happens at the end of a chromosome when the primer pops off?

Answer 35

there is a gap left that a telomerase fills.

Question 36

Telomerase

Answer 36

(a ribonucleoprotein) contains RNA which it uses to make several repeats of DNA to fill in the gap. WHICH means that the RNA is used as a template for DNA synthesis: REVERSE TRANSCRIPTION

Question 37

Telomere length and Aging/Imortality?

Answer 37

studies show that the somatic cells of individuals with progeria have shorter telomeres and do not divide as well in culture, but this is not proof that the disease is due to a decrease in a telomere length.