Bio 101: Chapter 16 Part 2
Meselson and Stahl
(late 1950s) devised clever experiment that distinguished btw the 3 replication models; their experiment supported semiconservative model,
Which models of replication were somewhat difficult to devise?
conservative and dispersion
leading strand
continuously synthesized DNA strand along template strand TOWARD the replication fork
Topoisomerase
helps relieve strain from untwisting by breaking, swiveling, and rejoining DNA strands
mismatch repair errors
hereditary defect in a mismatch repair enzyme allows cancer-causing errors to accumulate in the DNA faster than normal causing colon cancer
After 2 phosphates lost as a pyrophosphate, what happens immediately after?
hydrolysis of the pyrophosphate to 2 molecules of inorganic phosphate. makes up a coupled exergonic reaction driving polymerization of the complementary DNA strand being synthesized
important function of DNA repair enzymes
in skin cells to repair genetic damage caused by UV rays of sunlight
How do telomeres provide protective function?
postpone erosion of genes located near ends of DNA molecules but become shorter during every round of replication
replication fork
present at each end of replication bubble; Y-shaped region where parental strands of DNA are being unwound by SEVERAL proteins
Shortening of DNA does not occur in
prokaryotes bc of circular chromosomess
Synthesis of Lagging Strand Steps 1 to 4
1. Primase joins RNA nucleotides into a primer. 2. DNA pol III adds DNA nucleotides to primer, forming Okazaki fragment 1. 3. reaches next RNA primer to right, DNA pol III detaches. 4. Fragment 2 primed. DNA pol III adds DNA nucleotides, detaching when it reaches fragment 1 primer.
conservative model of replication
2 parental strands somehow come back together after process of replication (that is, the parental molecule is CONSERVED)
As each monomer joins the growing end of a DNA strand,
2 phosphate groups are lost as ONE MOLECULE of pyrophosphate
basic replication properties of DNA
2 strands are complementary, with stored info to reconstruct the other.
# nucleotide pairs in single chromosome E. Coli? time spent replicating E. Coli DNA into 2 identical daughter cells
4.6 million; less than 1 hour
Each human cell contains how many DNA molecules? # base pairs? time spent replicating all of a human cell's DNA? error frequency?
46 chromosomes (molecules) in nucleus; 6 billion base pairs; one cell takes just a few hours to copy all of this DNA with very few errors, only about 1 per 10 billion nucleotides. fast and usually very accurate
A new strand of DNA can elongate in which direction ONLY?
5' to 3'
Synthesis of Lagging Strand Steps 5 to 7
5. DNA pol I (different than pol III) replaces RNA nucleotides of adjacent primer with DNA nucleotides, adding to the 3' end of fragment 2. 6. DNA ligase forms bond btw newest DNA and DNA of fragment 1. 7. lagging strand in this region now complete.
2 DNA polymerases playing major roles in E.Coli DNA replication; # of DNA polymerases in eukaryotes
DNA Polymerase III and DNA Polymerase I; Eukaryotes more complicated with at least 11 DNA polymerases discovered so far
How do you elongate the other strand of DNA while obeying the 5 to 3 direction rule?
DNA pol III must work along the other template strand AWAY FROM replication fork
enzymes involved in filling the gap resulting from nuclease removing damage in with nucleotides using undamaged strand as a template are
DNA polymerase and DNA ligase
ATP vs. Nucleoside TP (Triphosphate)
NTP's sugar is deoxyribose. ATP's sugar is ribose. Like ATP, NTPs are chemically reactive partly bc triphosphate tails have an unstable cluster of negative charge
After parental strands separate....?
SINGLE-STRAND BINDING PROTEINS bind to unpaired DNA strands, keeping them from re-pairing
in each human telomere,
a 6 nucleotide sequence is repeated btw 100 to 1,000 times
short stretch of initial RNA nucleotide chain is called
a PRIMER and is synthesized by the enzyme PRIMASE
Requirements for DNA polymerases
a primer and a DNA template strand, along with complementary DNA nucleotides line up
According to Watson and Crick, when a cell copies DNA molecules, each strand serves as...; How do nucleotides line up along the template strand?
a template for ordering nucleotides into a new, complementary strand. line up according to base pairing rules and are linked to form the new strands, each an exact replica of the "parental molecule" of DNA in its place.
functions of telomeric DNA
acts as a kind of buffer zone protecting genes; also, specific proteins associated with it prevent staggered ends of daughter molecule from activating cell system's for monitoring DNA damage
DNA Polymerase III (E.Coli)
adds a DNA nucleotide to RNA primer and the continues adding DNA nucleotides, complementary to parental DNA template strand to the growing end of the new strand at an elongation rate of about 500 nucleotides per second in bacteria and 50 per second in humans
DNA ligase
an enzyme joining the sugar-phosphate backbones of all the okazaki fragments into a continuous DNA strand
Repairing incorrectly paired nucleotides due to DNA damage as well as replication errors take advantage of
base-paired structure of DNA
How does antiparallel arrangement of double helix affect replication?
bc of their structure, DNA polymerases can add nucleotides ONLY to the free 3' end of a primer or growing strand, NEVERRR to the 5' end
thymine dimers
cause the DNA to buckle and interfere with DNA replication; repairing this kind of damage prevents disorder xeroderma pigmentosum
xeroderma pigmentosum
caused by an inherited defect in nucleotide excision repair enzymes; hypersensitivity to sunlight and mutations in their skin cells from UV light left uncorrected, result in skin cancer
Meselson and Stahl experiment
cultured E. Coli in medium containing nucleotide precursors labeled with heavy nitrogen isotope N15. transferred bacteria to medium with N14. sample taken after DNA replicated once, then twice. DNA extracted from sample bacteria. Then, they centrifuged each DNA sample to separate DNA of different densities.
Lagging strand
discontinuously synthesized DNA that elongates as a series of segments called Okazaki fragments AWAY FROM replication fork
Watson and Crick's hypothesis for how DNA replicates (in a 2nd paper)
double helix is pair of templates, complementary to one another; before duplication, hydrogen bonds btw bases broken & 2 chains unwind and separate; each acts as template for formation of new partner chain onto itself, eventually 2 pairs of chains, where we only had one before. Moreover, the sequence of pairs of bases will have been duplicated exactly.
telomerase
enzyme that catalyzes lengthening of telomeres in eukaryotic germ cells (sex cells)
nuclease
enzyme that cuts out certain damaged segment of DNA or RNA
primase
enzyme that starts a complementary RNA chain from a single RNA nucleotide, adding RNA nucleotides one at a time, using the parental DNA strand as a template. completed primer, 5-10 nucleotides long, is thus base-paired to template strand.
DNA polymerases
enzymes that catalyze synthesis of new DNA by adding nucleotides to preexisting chain
helicases
enzymes that untwist the double helix at replication forks, separating 2 parental strands, making them available as template strands
Results/Conclusion of Meselson & Stahl Experiment
first replication in N14 produced band of hybrid 15-14 DNA which eliminates conservative model. 2nd replication produced both N14 & hybrid 15-14 DNA, which eliminates the dispersive model and supports the semiconservative model . CONCLUSION: DNA replication is semiconservative
Dispersive model of replication
following replication, all 4 strands of DNA have a mixture of old and new DNA (almost like patches of old and patches of new DNA on the same strand)
Where do the nucleotides being added to the growing DNA strand come from?
from a nucleoside triphosphate( nucleoside, sugar + base, with 3 phosphate groups) supplies adenine nucleotide to DNA
Where will the new DNA strand start from?
from the 3' end of the RNA primer
What scientists have learned about eukaryotic DNA replication suggests that most of the process is
fundamentally similar for prokaryotes AND eukaryotes.
large tumors contain very short telomeres so eventually..
further telomeric shortening will result in self-destruction of the tumor cells
Prediction about DNA in Watson and Crick's first published paper
it hasn't escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material
What happens if DNA polymerase finds an incorrectly paired nucleotide?
it removes the nucleotide and then resumes synthesis (it deletes the wrong one and enters the new one)
Where did the initial nucleotide chain produced during DNA synthesis come from?
its actually a short stretch of RNA, not DNA
repair of damaged DNA so important to the survival of an organism that...
its no surprise that there are many different DNA repair enzymes that have evolved. 130 identified in humans and 100 in e.coli so far
Nucleotide alteration AFTER replication
maintenance of genetic info coded in DNA requires frequent repair of various kinds of damage. constantly subjected to harmful chemical and physical agents;
Origins of eukaryotic DNA molecule (chromosome)
may have hundreds or thousands of replication origins. multiple bubbles form and eventually fuse to speed up the replication
shortening of telomeres provides protection too?
may protect against cancer by limiting number of divisions that somatic cells can undergo
telomeres contain ... instead of ....
multiple repetitions of one short nucleotide sequence instead of genes
Okazaki fragments
named after Japanese scientist that discovered them; fragments of the lagging strand that are about 1,000 to 2,000 nucleotides long in e.coli and 100-200 in eukaryotes
complications of replication in 5 to 3 direction only
once primer is removed at very end of template strand it cannot be replaced with DNA bc there is no 3' end available for nucleotide addition. leads to repeated rounds of shorter and shorter DNA molecules with uneven ends
rate of initial pairing errors btw incoming nucleotides and those in the template strand
one in 100,000 nucleotides even though the DNA polymerases proofread each nucleotide against its template as soon as it is added to the growing strand.
amount of primers required for DNA pol III to make leading strand; lagging strand
only ONE; each Okazaki fragment must be primed SEPARATELY
origins of replication
particular site or sites where replication of DNA molecule begins, consists of short stretches of DNA having a SPECIFIC sequence of nucleotides (E. Coli-circular chromosome with a SINGLE origin)
mutations
permanent changes in DNA perpetuated through successive replications
Initiation of DNA replication
proteins initiate process that recognize this specific sequence and attach to DNA, separating strands & opening up a replication "bubble". from there, replication proceeds in both directions from origin until entire molecule is copied.
If DNA pol III is synthesizing the complementaryt strand continuously by elongating the new DNA in the 5 to 3 direction, where is the DNA pol III located during this time?
remains in the replication fork on template strand and continuously adds nucleotides to new complementary strand as the fork progresses.
Nucleotide excision repair
removes and replaces segment of damage DNA from a strand using undamaged strand as the template for new one
telomeres
repetitive DNA at ends of eukaryotic chromosomes DNA molecule. structures that protect organism's genes from being eroded during successive rounds of replication
Staggered ends of a DNA molecule, which often result from double-stranded breaks, can trigger
signal transduction pathways leading to cell cycle arrest or CELL DEATH
telomere length
tends to be shorter in older individuals
Antiparallel qualities of DNA strand
the ends of strand are different, each with directionality; antiparallel meaning oriented in OPPOSITE directions to each other like a highway, so 2 NEW strands formed during replication must also be antiparallel to their template strands
Even though the unwound parental DNA templates are available for synthesis of a complementary strand....
the enzymes that synthesize DNA CANNOT initiate synthesis of a polynucleotide, but only add nucleotides to the end of an already existing chain that is base-paired with the template strand
Untwisting of double helix causes
tighter twisting and strain ahead of replication fork
Watson and Crick's "copying mechanism" in DNA replication is analogous to ...
to using a photographic negative to make a positive
mismatch repair
uses specific enzymes to remove and replace incorrectly paired nucleotides that have evaded DNA polymerase proofreading; result from replication errors
Why would representing the DNA polymerase molecules as locomotives moving along a "railroad track" be an inaccurate model?
various proteins that contribute form a single "DNA replication machine". 2nd DNA replication may not move along DNA, rather, the DNA may move through the complex during the replication process
What does Watson and Crick's model of DNA replication predict? their model described as?
when a double helix replicates, each daughter molecule will have one old strand from parental molecule and one newly made strand; semiconservative model
When does primase act as a "brake" in replication?
when interacting with proteins at the fork, slowing the progress of the replication fork and coordinating the placement of primers and rates of replication on the leading and lagging strands
