lecture 6 & 7- DNA replication I, II Flashcards
without DNA replication….
cannot have transfer of into from parent to daughter cell
in eukaryotes, DNA replication happens in ___ phase
S
DNA replication is the duplication of the _____, in which the stored genomic information is handed down to the next generation
cellular genome
what are the 3 alternative hypotheses for DNA replication models
1- semi-conservative
2- conservative
3- dispersive
Meselson and Stahl demonstrated that replication is ___, meaning ___
semi-conservative
one old strand from parents and one new daughter strand
describe Meselson and Stahl’s experiment
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)
describe beginning of DNA replication in vivo
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)
at the replication fork, the DNA of both new daughter strands is synthesized by a multienzyme complex that contains the ____
DNA polymerase
replication is initiated at ___ and proceeds ___
origins
bidirectionally
bacterial chromosome: theta-form replication
- 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
eukaryotic chromosomes for replication
- 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
replication is ___, only one daughter strand is synthesized continuously, the other is made as a series of discontinuous fragments, ____
semidiscontinuous
Okazaki fragments (1-2 kb long in bacteria; 100-200 nucleotides in eukaryotes)
all DNA polymerases require a ___ and a ____
template strand & primer
DNA polymerases require a primer to provide a ___ to add new nucleotides (for extension)
3’ OH
DNA polymerases elongate DNA in the _____ direction
5’ to 3’
many DNA polymerases have DNA Exonuclease activity (3’ to 5’), which functions in ___
proofreading
polymerase and nuclease activity reside in ___
different sites
chemistry of polymerization
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
DNA polymerase structure
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)
the accuracy of the polymerase functions at the level of ___
shape recognition
(shape of active site in open and closed conformations)
the DNA polymerase active site contains ____ that are required for catalysis
two divalent metal ions (cofactors)
Mg2+ deprotonates the primer 3’OH and binds the incoming dNTP and facilitates departure of the pyrophosphate
in the DNA polymerase structure, formation of a phosphodiester bond leads to opening of the ___ and movement of ___ by one base pair
fingers
template/primer
DNA slides down to exonuclease active site for removal of mismatched nucleotides
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
DNA polymerase accuracy is due to 3 things:
1- nucleotide selection by DNA polymerase (shape recognition in active site)
2- proofreading
3- mismatch repair
primase initiates on ___ containing a specific ___, activity increases when associated with ___
ssDNA
primer
helicase
SSB’s do what?
stabilize ssDNA prior to replication
they use cooperative binding to ensure quick coverage of exposed template
DNA polymerase moves along the template strand in the ___ direction and adds nucleotides to the ___ of the newly synthesized strand
3’ to 5’
3’
a new DNA strand elongates only in the 5’ to 3’ direction because…
DNA polymerase can only add nucleotides to the free 3’ end
