THEME 4 MOD 2 Flashcards
What did francis crick, james watson and Rosalind franklin discover about the structure of dna
helical structure with purines paired with pyrimidines
Watson and Crick og statement on dna replication
It has not escaped our notice that the specific pairing that we have postulated immediately suggests a copying mechanism for genetic material
Watson and crick 1954 hypothesis for dna replication
Dna is a pair of complementary template chains where h bonds must be broken between the chains for unwinding, seperation and replication to occur
What is meant by a complementary strand in terms of dna replication
each strand contains the info necessary to reconstruct the other
each strand serves as the template for the ordering of new nucleotides based on the base-pairing rules
semiconservative model of dna replication (watson and crick)
starting with one dna molecule, the two synthesized daughter strands would have one parental and one newly synthesized strand of dna
Who proved that dna replicates in a semiconservative manner
Matthew Meselson and Franklin Stahl
using e coli bacteria cells over multiple generations:
first grew ecoli in medium with 15 N isotope, then subsequently switched the ecoli to 14 N medium
They then extracted dna from the growing bacteria and centrifuged it through a solution that would separate different densities of radioactively labeled dna.
The 15 iostope makes the dna heavier
bacteria growing in 15 n strand had one distinct band
bacteria after one round in 14 n: a different distinct band (hybrid of 14N and 15N)
could reject conservative model because there was no distinct 2nd 15 N band
Then left with either semiconservative or dispersive model
after continual rounds of replication:
one distinct gel band of 14 N dna and one distinct band of 14 N and 15N dna
confirmed each new round of dna had one old and one new strand
Importance of fluorescent nucleotides in understanding dna replication
can label individual nucleotides
rounds of replication of eukaryotic cells in media with fluorescent nucleotides preformed: found hybrid and fully labelled nucleotides in chromosomes (some faint, some darkly fluorescing strands)
confirmed semiconservative mechanism of DNA was also in eukaryotic cells
origin of replication in prokaryotes
One orgin of replication, replication occurs along circular chromosome from one origin of repliaction.
process of dna replication
similar to transcription
template copied from 3’ to 5’ end, synthesizes daughter strand from 5’ to 3’ direction.
RNA primer synthesized complementary to template strand
enzymatic machinery (DNA polymerase) can synthesize daughter strand once the 5-10 nucleotide primer annealed to the template strand
incoming complementary nucleotide forms hydrogen bond with base pair and forms phosphodiester bond with 3’ hydroxyl on elongating polymer (daughter strand), producing pyrophosphate in the process.
This polymerization of each incoming nucleotide is catalyzed by DNA polymerase enzyme (in 5’ to 3’ dir)
Unwinding of DNA
separate parental strands at replication forks
Leading vs lagging strand
replication of leading strand is continuous from the RNA primer, DNA polymerase continuously adds nucleotides to strand as replication fork progresses.
The lagging strand is synthesised in discontinuous fragments called Okazaki fragments (Reiji Okazaki) formed from separate primers. Strand synthesized in direction away from replication fork
Strand is lagging because a certain amount of nucleotides of the template strand need to be revealed to synthesize the Okazaki fragment
Lagging strand needs post replication processing: after dna polymeraze forms an okazaki fragment, another dna polymerase will replace the primer with dna nucleotides
initiation proteins, replication complex
Dna helicase: bind to parental strands at orgin of replication and initiate unwinding, break hydrogen bonds between base pairs
single stranded binding proteins: Bind to parent strands to stabilize them so they don’t rejoin each other until elongation begins
Topoisomerases: bind upstream of replication fork, minimize torsional strain
What marks the begininning of dna synthesis?
primer synthesis
rna polymerase
synthesizes rna primers
DNA poly I VS III
DNA polymerase III: elongation of daughter strand
DNA polymerase I: replace rna primers with dna nucleotides between Okazaki fragments
Do eukaryotes and prokaryotes use the same set of DNA polymerases?
No:
prokaryotes: rna primer is excised and replaced during replication on lagging strand
eukaryotes: lagging fragments have 3’ free phosphate of their sugar phosphate backbone
- dna ligase synthesizes a phosphodiester bond between the 3’ free phosphate end of one okazaki fragment with the 5’ end the adjacent okazaki fragment along the backbone.
improper base pairing, errors in DNA replication
- improper initial nucleotide base pairing:
DNA polymerase proofreading mechanism proofreads each nucleotide in correspondence with the template strand as its added to the daughter strand.
if there is an incorrect pairing dna polymerase removes the improper nucleotide and replaces it with the correct one, then continues with replication
similarities and differences of replication in eukaryotes vs prokaryotes
differences:
- prokaryotes: one origin of replication where synthesis then occurs along the circular chromosome.
Eukaryotes:
- multiple origins of replication, replication occurs along linear chromosomes
similarities:
-require primer for intiation of replication
- synthesize daughter strands in 5’ to 3’ direction
-have leading and lagging strands
-replication complex with specialized proteins
What is unique about the 5’ end of a daughter strand in eukaryotes
- because prokaryotes are circular, once replication is complete the 5’ end can form a phosphodiester bond with the 3’ end of the daughter strand
- eukaryotes cannot do this as linear chromosomes, and once the rna primer is removed there is no 3’ end to synthesize the additions of new nucleotides and phosphodiester bond with the 5’ end of the daughter strand
What is the problem with an exposed 5’ end of a daughter strand
Through rounds of replication the DNA strands will shorten and new daughter strands will have shortened and uneven ends, and genes will erode away
What is present in eukaryotic daughter strands to protect the integrity of synthesized dna throughout cycles of replication?
- repeating short sequences called a telomere is added to the 5’ end of the strand to act as a buffer zone to protect the coding region of dna, however these telomeres become shorter as people age
- humans contain a telomere sequence of nucleotides: TTAGGG repeated over and over 100s to thousands of times, creating tandem repeat of G-T rich sequences
Why would it be dangerous for telomeres to shorten with time in certain cells
telomere length must be maintained in cells whos genome must be passed on intact to subsequent generations
in gametes, shortened telomeres could mean missing information in sex cells
in embryonic stem cells shortening telomeres could occur more frequently due to series of replication
telomere shortening doesn’t happen in these cells! telomerase enzyme catalyzes lenthening of telomeres
How does telomerase work?
its a reverse transcriptase that synthesizes dna from an rna template, which is built into the complex of this ribonucleoprotein
attaches to the telomere end of the chromosome and adds telomere repeats to the template strand. Dna polymerase, primase and ligase can then go in and synthesize the telomere repeats for the daughter strand.
