EXAM 4 - Chapter 11b, Replication of DNA Flashcards
________ possible modes of replication were suggested by its structure
three
1: Conservative Replication suggests that…
two old strands come back together after replication, entire old molecule conserved in one of the two molecules
unwraps and makes new, 1 strand to two strands, entire old molecule conserved
2 Semiconservative Replication suggests that…
One new and one old strand form a new molecule. half of the old molecule is conserved in each of the new molecules
two old strands come apart, two new and half old half new
3 Dispersive replication suggests that…
a patch work of new and old forms the new molecules, old strand is dispersed in new strands
Mix of old and new strands
_____________ were the first to demonstrate ________ DNA replication
Meselson and Stahl, Semiconservative
Meselson and Stahl showed semiconservative using
density gradient centrifugation
What they did was…
Grew bacteria in heavy N15 and then transferred them to normal N14
they put gel inside test tube and dropped molecules in and spin it really fast, gel is porous and going to travel based on weight
After one generation in N14 medium,
all DNA was half in N15 and half in N14, ruling out conservative replication
After two generations,
half was N14/N14(light) and half was N15/N15(int.), ruling out dispersive replication
Conservative replication would have been
half in DNA heavy and 1/2 in DNA light
Dispersive would have been..
all the same weight
DNA replication occurs when…
two strands open out, new nucleotides line up opposite the old complementary bases, and DNA Polymerase catalyzes their addition to the new strand
DNA Polymerase is the
enzyme
DNA polymerases are…
large molecules
Most cells have…
multiple polymerases (human cells have at least 15)
Only one or two of these…
replicate chromosomes, the rest are involved in DNA repairs
There are ____ steps for DNA Replication
10
DNA Replication Step #1
Helicase initiates replication by denaturing (unzipping) DNA using energy from ATP
Denatures H bonds between nitrogenous bases
DNA replication ALWAYS happens from…
right to left
Where the DNA is open during replication is called
the fork
DNA Replication Step #2
Topoisomerase travels in front of helicase, relieving the supercoiling cause by Helicase
if no topo, helicase would push it so hard it would get bunched up
DNA Replication Step #3
Single Strand Binding proteins coat single stranded DNA to keep it from renaturing (reforming double helix)
If proteins werent there, strands would just come back together
DNA Replication Step #4
Because of base-pairing relationships, nucleoside triphosphates tine up across from complementary bases in old strand, which is the template for the new strand
DNA Replication Step #5
DNA Polymerase III splits off last 2 high energy phosphates (one at a time), polymerizing the nucleotide into the new DNA strand
making DNA polymers our of monomers, when the phosphates are broken off, able to be glued together
DNA polymerase III has ____ limitations
two
The two limitations of DNA polymerase are
- it cant start strands
- It can’t add new nucleotides going toward the 5’ end of the growing DNA chains (can only add going toward 3’ end OF THE NEW STRAND)
BUILDS 5 TO 3
_______ overcomes first limitation of DNA Polymerase III
Primase
DNA Replication Step #6
Primase replicates the first 12-20 nucleotides with RNA (making the RNA primer chain) on which DNA Polymerase III can then build
DNA Replication Step #7
Synthesis of leading strand
since strand run in the 3’ to 5’ direction (same as fork), so new strand is build continuously after a primer strand is produced
keep in mind template strand is from 3’ to 5’, so that means strand that is being build is 5’ to 3’)
(#8)__________ used to overcome second limitation of DNA Polymerase III which is…
Okazaki fragments….it can only add toward the 3’ end of growing chain
(#8)This second limitation is overcome by…
building the lagging strand in a discontinuous fashion using Okazaki fragments
DNA Replication Step #8
Synthesis of lagging strand
(#8) Template strand runs from 5 to 3 direction, so if new strand built continuously…
new strand would be built in opposite direction since DNA polymerase can only synthesize from 5 to 3 direction
Bottom strand is in a way technically running from….
left to right
(#8) SO lagging strands built…
discontinuously in fragments (Okazaki fragments) each built in 5 to 3 direction, but OVERALL GOING IN 3 TO 5 (same as fork)
they jump backwards then build another then jump backwards and then building
(#8) its lagging strand so its…
a little slower, since have to jump backwards
(#8) After the frist okazaki fragment is built…
another primer then a fragment is built, each fragment built in the 5 to 3 direction, but are laid down in overall 3 to 5 (same as fork)
kind of like backwards leap frog, after making one enzyme has to jump backwards to make the next
(#8) each fragment is made…then..
individually and discontinuously and then connected to others
DNA Replication Step #9
DNA Ligase connects okazaki fragments by closing the last gap between fragments
**dont need this on leading/template strand
DNA Replication Step #10
DNA Polymerase I (both strands) follow replication fork, replacing the primer strand with DNA and correctly errors when found
must switch out the RNA with DNA, also proofreading and correctly errors
Telomeres are
the ends of eukaryotic chromosomes
Telomeres have…
special repeating sequences that prevent the end of chrmosomes form attaching to one another
Replication of telomeres is problematic since…
replication of lagging strand does not start at very end of chromosome
cant start copying at end, cant grab onto very end
replication does not start at end because…
of limitations of DNA Polymerase III
As a result, telomeres and whole chromosome…
may shorten over many round of replications
As they get shorter, it starts affecting functions since you have less of each one
Shortened telomeres have been implicated in…
aging
Some cells including cancer cells have…
enzyme Telomerase which lengthens telomeres
Telomerase attaches to…
3’ end of DNA strand
Interal RNA template used to…
line up DNA nucleotides and extend the 3’ end of the chromosome
After the 3’ end is lengthened…
it can be long enough for DNA polymerase to attach and produce okazaki fragments (primase -> RNA primer 1st)
Okazaki fragment extends bottom strand in 5 to 3
Now entire chromosome is longer
**lengthens chromosome on both ends THEN builds okazaki fragments
Molecular structure of prokaryotic chromosomes
- circular double strands of DNA with both ends connected
- proteins DO NOT regularly and permanently attach to them
- there is typically one main chromosome in a cell, sometimes with “extra” small circular chromosomes called PLASMID
Molecular structure of eukaryotic chromosomes
- consist of both DNA and proteins
- many of the proteins are histones which form nucleosomes, around which the DNA is wrapped in a groove
- two copies of each of four histones compose the nucleosomes
- they contain many positively charged peptides to attract and bind DNA
Histones…
help DNA coil and super condensed so that it can fit a lot of DNA
Histones have…
two of each four histones which are H2A, H2B, H3, H4
5th histone HI…
binds linker DNA which occurs between the DNA segments wrapped around nucleosomes
DNA wrapped around nucleosomes resembles…
beads on a string
Adjacent nucleosomes are..
organized into a thicker fiber, 30 nm in diameter
DNA wrapped around core, then those cores stacked tightly on top of each other
The 30 nm fiber of each chromosome is …
attached to a filamentous network of proteins termed the Nuclear Matrix
The matrix consists of..
protein fibers which line the inner nuclear membrane (nuclear lamina) and other fibers stretching throughout the nucleus
Other proteins connect..
regions of DNA to the Nuclear Matrix
these other proteins also…
bend DNA into radial loop domains containing 25,000-200,000 necleotide base pairs
DNA and chromosomes they make up are thus…
anchored to specific regions in the nucleus
Chromosomes are…
NOT free to move within the nucleus, but are restricted to specific regions by binding to the nuclear matrix
During cell division…
chromosomes condense into thicker, shorter structures by a series of loops and bends so they can be moved without tangling or breaking the long DNA fibers
takes lot of energy to hold this shape
