Lecture 14

Question 1

7 traits of replication in euks and proks

Answer 1

1. complementary base pairing
2. semi-conservative
3. unwinds the helix and moves as fork
4. bidirectional
5. requires RNA primer
6. discontinuous on lagging strand
7. requires enzymes called DNA polymerase

Question 2

Origin

Answer 2

sequence of DNA where replication is initiated

Question 3

Single well defined sequence that serves as the origin (only one for the entire chromosome)

Answer 3

OriC

Question 4

Where are eukaryotic origins found?

Answer 4

more variable (many on each chromosome)
Location and sequence has been determined by genetic and molecular approaches

Question 5

Replicon

Answer 5

unit of genome in which DNA is replicated. Each contains an origin for initiation of replication.

Question 6

Bidirectional

Answer 6

Replication fork moves in two directions

Question 7

Unidirectional

Answer 7

Replication fork moves one way

Question 8

Replicon timing

Answer 8

they do not all start at the same time (early vs late replication)

Question 9

How can we map eukaryotic origins?

Answer 9

2 Dimensional gels

Question 10

1st dimension

Answer 10

Separates on size with electric current

Question 11

2nd dimension

Answer 11

exaggerates shape due to high agarose % and ethidium bromide

Question 12

How do we extract the DNA from the gel?

Answer 12

Southern blot with hybridization

Question 13

ARS

Answer 13

yeast Autonomously Replicating Sequences

Question 14

ARS’ contain _____ sequences

Answer 14

CIS acting

Question 15

ARS” confer ______ to plasmids

Answer 15

autonomous replication

Question 16

An ARS is a well defined ________

Answer 16

eukaryotic origin

Question 17

ARS interracts with the protein complex known as..

Answer 17

ORC, origin replication complex

Question 18

What type of protein does ORC have?

Answer 18

Trans acting protein

Question 19

How do they find ARS’s

Answer 19

mutagenesis

Question 20

Linker Scanning Mutagenesis

Answer 20

Disrupts CIS acting sequence but maintains SPACING

Question 21

How many bp elements are found in ARS I

Answer 21

4 related 11 bp elements important for origin function

Question 22

Element A contains…

Answer 22

ARS consensus sequence of 11 bp and is absolutely essential for origin function

Question 23

Domain of DNA that is easily unwound

Answer 23

B domain (likely A-T rich)

Question 24

What is an ORC?

Answer 24

a complex of 6 proteins that bind to an ARS

Question 25

What does ORC do?

Answer 25

facilitates local unwinding at the origin with helicase activity

Question 26

Where does the ORC sit?

Answer 26

on the ARS throughout the cell cycle until activated

Question 27

What activates the ORC?

Answer 27

CDC 6

Question 28

Proposed model of the ORC and ARS

Answer 28

B region unwinds, cell cycle trigger

Question 29

player 1 after orc activation and CDC6

Answer 29

1. RPA cellular replication protein A

Question 30

What does RPA do?

Answer 30

3 subunit ss DNA binding protein

Also binds pol alpha

Question 31

Player 2 after orc activation and CDC6

Answer 31

DNA pol alpha/primase

Question 32

What does DNA pol alpha/primase do?

Answer 32

synthesizes 1st RNA primer

Synthesizes DNA on lagging strand

Question 33

Player 3 after Orc activation and CDC6

Answer 33

3. RFC, replication factor C

Question 34

What does replication factor C do?

Answer 34

Binds 3’ end of nascent DNA strand

Question 35

Player 4 after orc activation and CDC 6

Answer 35

4. PCNA, proliferating cell nuclear antigen

Question 36

What does PCNA do?

Answer 36

subunit of DNA pol delta