replication 1

Question 1

what phase of cell cycle is DNA replication

Answer 1

s-phase

Question 2

what is the name of the region of a chromosome/plasmid were replication initiates

Answer 2

origin (ori)

Question 3

how many origins of replication do prokaryotes and eukaryotes have?

Answer 3

prokaryotes have one

eurkarytoes have multiple/chromosome

Question 4

what is a replicon

Answer 4

a unit of DNA that is replicated from one origin of replication

Question 5

how often/cell cycle does a replicon work?

Answer 5

once

Question 6

depending on cell type and age human chromosomes have how many replicons?

Answer 6

10,000 - 100,000

Question 7

BrdU is an analog to what

Answer 7

thymine

Question 8

what is a foci

Answer 8

an area of DNA with up to several hundred replication forks

Question 9

define semiconservative DNA replication

Answer 9

Each strand acts as a template for the synthesis of a new DNA molecule by the sequential addition of complementary base pairs, generating a complementary base sequence

Question 10

how was it discovered DNA replication forks are bidirectional?

Answer 10

cells were grown in lightly radiolabeled thymine to lightly label all DNA then for a brief time grown in heavily labeled thymine to heavy label it, the rep fork was dense, chromosomes were imaged and label was seen equally at both replication forks

Question 11

exception to bidirectional replication?

Answer 11

colE1 plasmid in e. coli. was discover by electron microscopy of restriction enzyme treated plasmid, after linearization the rep bubble was same distance from cut each time

Question 12

all DNA polymerases make DNA in what direction

Answer 12

5’ –> 3’

Question 13

describe pulse chase experiment that discovered okazaki fragmentss

Answer 13

e.coli were infected with a bacteriaphage, the phage duplicating, while duplicating different times pulses of radioactivity, the shorter pulses = shorter DNA fragment, = okazaki fragments

Question 14

what is a primer that allows DNA synthesis adding?

Answer 14

a 3’-OH

nick in preceding DNA, and RNA primer, or a DNA binding protein with covalent nucleotide link can serve as primers

Question 15

most common replication structure in bacteria, what type of primer?

Answer 15

theta mode, RNA

Question 16

explain rolling circle replication

Answer 16

circular DNA, on stand nicked, 3’ end is extended/5’ end is displaced

Question 17

DNA polymerase I in e.coli 3 distinct activities, what are they/what do they do

Answer 17

5’->3’ polymerase replaces removed primer with new DNA (nick translation)
3’->5’ exonuclease proofreading (“klenow, finger/palm)
5’->3’ exonuclease removes RNA primer

Question 18

why was studying DNA polymerase III done in a conditional mutant?

Answer 18

zero cell proliferation if no DNA poly III, needed to study under restrictive and permissive conditions

Question 19

important subunits of DNA polymerase III holoenzyme (3)

Answer 19

alpha, DNA polymerase
epsilon, 3’ -> 5’ exonuclease
beta, sliding clamp

Question 20

what protein is the initiator of chromosomal replication?

Answer 20

DNA A

Question 21

what is the motif that is recognized to begin e.coli replication

Answer 21

DNA A box in the oriC

Question 22

throughout the cell cycle/prior to replication initiation what proteins are bound to e.coli ORC?

Answer 22

the high affinity DnaA boxes R1, R2, R3 bound by DnaA and Fis (repressor) binds inbetween

Question 23

what proteins bind do ORC during replication initiation of e.coli to form prereplication complex

Answer 23

DnaA is able to bind moderate and low affinity DnaA boxes (tau and I sites)

Fis (repressor) is displaced by IHF

Question 24

How is DNA unwound by prereplication complex in e.coli?

Answer 24

tortional stress from DnaA protein monomers

Question 25

name of protein that binds DnaA to accelerate/coordinate cell cycle initiation

Answer 25

DiaA (DnaA initiator associating protein)

Question 26

after unwinding of AT-rich oriC region DnaA recuits what

Answer 26

two DnaB-DnaC complex

B = helicase (hexameric, unwind DNA at rep fork)

C = helicase loader, bring DnaB to the DNA then dissociates

Question 27

name of protein that bind DNA after unwinding and it’s purpose

Answer 27

single strand binding protein (SSB) prevents reannealing

Question 28

what are the proteins recruited by DnaB to make RNA primers

Answer 28

DnaG primase and DNA pol III holoenzyme (extends primers)

Question 29

how does methylation prevent over replication of ecoli DNA

Answer 29

Dam (DNA adenine methyltransferase) methylates adenines in a palindromic OriC sequence, the protein SeqA binds the hemimethylated areas and inhibits formation of new initiation complexes

as DnaA poorly recognizes hemimethylated DNA can’t initiate replication

Question 30

How does dnaA regulate itself

Answer 30

DnaA protein functions as its own repressor

after chromosomes double DnaA will be sequestered elsewhere in the genome