lecture 6 & 7- DNA replication I, II

Question 1

without DNA replication….

Answer 1

cannot have transfer of into from parent to daughter cell

Question 2

in eukaryotes, DNA replication happens in ___ phase

Answer 2

S

Question 3

DNA replication is the duplication of the _____, in which the stored genomic information is handed down to the next generation

Answer 3

cellular genome

Question 4

what are the 3 alternative hypotheses for DNA replication models

Answer 4

1- semi-conservative
2- conservative
3- dispersive

Question 5

Meselson and Stahl demonstrated that replication is ___, meaning ___

Answer 5

semi-conservative
one old strand from parents and one new daughter strand

Question 6

describe Meselson and Stahl’s experiment

Answer 6

used 2 isotypes of N to change DNA density (15 N=heavy 14N=light)
- grew bacteria in media containing only 15N for many generations
- transferred bacteria to 14N media
- extracted DNA from several generations of bacteria
- after density gradient centrifugation, looked at bands:
. after 1st generation, conservative eliminated (one hybrid of 15N/14N band)
. after 2nd generation, dispersive eliminated (one 14N and one hybrid)

Question 7

describe beginning of DNA replication in vivo

Answer 7

replicon- a unit of genome in which DNA is replicated
-once replication starts at origin, no way to go back/stop
- 1st step of replication= separation of DNA strands at replication eye, initiates replication
.replication fork inside replication eye, fork has all the enzymes needed for replication
. most common- replication forks inside eye moving both sides (BIDIRECTIONAL)

Question 8

at the replication fork, the DNA of both new daughter strands is synthesized by a multienzyme complex that contains the ____

Answer 8

DNA polymerase

Question 9

replication is initiated at ___ and proceeds ___

Answer 9

origins
bidirectionally

Question 10

bacterial chromosome: theta-form replication

Answer 10

• bacteria have singular circular chromosome, hydrogen bonds keep DNA helix together (no nucleosome)
• origin- any DNA can bind to (general feature: A-T rich sequence)
• E. coli: single origin, OriC (245 bp sequence)
  .30 diff proteins required for E. coli replication
  . rate: 1,000 nucleotides per second

Question 11

eukaryotic chromosomes for replication

Answer 11

• each chromosome composed of many replicons
• many origins necessary because slower replication and more DNA present (100 nucleotides per second)
• ARS elements from yeast equivalent to OriC for E. coli (any sequence containing ARS in yeast can be replicated)

the start of S phase activates the first replicon, not all replicons are activated at the same time
. all of the DNA must be replicated only once prior to cell division

Question 12

replication is ___, only one daughter strand is synthesized continuously, the other is made as a series of discontinuous fragments, ____

Answer 12

semidiscontinuous

Okazaki fragments (1-2 kb long in bacteria; 100-200 nucleotides in eukaryotes)

Question 13

all DNA polymerases require a ___ and a ____

Answer 13

template strand & primer

Question 14

DNA polymerases require a primer to provide a ___ to add new nucleotides (for extension)

Answer 14

3’ OH

Question 15

DNA polymerases elongate DNA in the _____ direction

Answer 15

5’ to 3’

Question 16

many DNA polymerases have DNA Exonuclease activity (3’ to 5’), which functions in ___

Answer 16

proofreading

Question 17

polymerase and nuclease activity reside in ___

Answer 17

different sites

Question 18

chemistry of polymerization

Answer 18

1- new DNA is synthesized from dNTP’s
2- in replication, 3’ OH group of the last nucleotide on the strand attacks the 5’ phosphate group of incoming dNTP
3- two phosphates cleaved off
4- phosphodiester bond forms between the 2 nucleotides
5- and phosphate ions are released

Question 19

DNA polymerase structure

Answer 19

template & primer
fingers (dNTP-binding site) dNTP enters between thumb and fingers
thumb
palm (polymerase active site)

base pairing in template causes fingers to close over, positioning substrates in the catalytic site (in the palm)

Question 20

the accuracy of the polymerase functions at the level of ___

Answer 20

shape recognition

(shape of active site in open and closed conformations)

Question 21

the DNA polymerase active site contains ____ that are required for catalysis

Answer 21

two divalent metal ions (cofactors)

Mg2+ deprotonates the primer 3’OH and binds the incoming dNTP and facilitates departure of the pyrophosphate

Question 22

in the DNA polymerase structure, formation of a phosphodiester bond leads to opening of the ___ and movement of ___ by one base pair

Answer 22

fingers

template/primer

Question 23

DNA slides down to exonuclease active site for removal of mismatched nucleotides

Answer 23

the slow incorporation yet rapid removal of mispaired dNTP underlies accuracy of DNA polymerase

• now, correctly paired DNA slides back to polymerase active site and DNA synthesis can be resumed

Question 24

DNA polymerase accuracy is due to 3 things:

Answer 24

1- nucleotide selection by DNA polymerase (shape recognition in active site)
2- proofreading
3- mismatch repair

Question 25

primase initiates on ___ containing a specific ___, activity increases when associated with ___

Answer 25

ssDNA
primer
helicase

Question 26

SSB’s do what?

Answer 26

stabilize ssDNA prior to replication

they use cooperative binding to ensure quick coverage of exposed template

Question 27

DNA polymerase moves along the template strand in the ___ direction and adds nucleotides to the ___ of the newly synthesized strand

Answer 27

3’ to 5’
3’

Question 28

a new DNA strand elongates only in the 5’ to 3’ direction because…

Answer 28

DNA polymerase can only add nucleotides to the free 3’ end