CBG.03 Flashcards
How many origins of repliaction are oon a prokaryotic chromsome
one
How can you recognize an origin of replication
A-T rich
What is the job of initiator proteins in DNA replication
allow the DNA to melt below 90 degrees
What is the function of helicase in DNA replication
continues melting using ATP
What three things must replication acheive
Must copy the information with a high fidelity
must be able to melt the double helix and sythesise new DNA in both the 3-5 and 5-3 simulatenously
Must be able to carry this out without getting into a tangle
How many origins of repliaction do Eukaryotes have in each chromosome?
More than one, humans have about 50,000 origins of replication
What did Meselson Stahl discover?
By using density gradient centrifugation and E.coli grown on media with different isoptopes of Nitrogen worked out thatDNA is replicated semi-conservatively by DNAP
Explain the role of the Klenow fragment
The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin. the klenow fragment has two active sites, a polymerase site 5’-3’, which makes the new DNA, and an exonuclease site 3’-5’, which is used for proof reading and removes nucleotides which are wrong, cleaves them off and reduces the error rate don to 10^-6
DNAP-1
is the priciple repair polymerase in E.coli, it patches in the holes and is needed in quite a large number in order to scout for holes, and therefore requires a high copy number
What different types of mutation can arise from transient base tautomery?
If a base is in the wrong tautomeric form during replication it can result in a base pair mismatch, called a lesion, which unless corrected will become a mutation. All four bases are capable of either amino -imino or keto-enol tautomery. Commonly (amino)cytosine:Guanine base pair, and imino-cytosine : adenine also base pair
also with small changes to helix geometry G and T are able to pair with 2 hydrogen bonds
Why does DNAP have to be unidirectional?
because durin
what is the structure of a replicating fork?
Assymetric
How long is a typical Ozaki fragment
Prokaryotes - 1000 - 2000 nucleotides
Eukaryotes - 100 -200 nucleotides
DNA primase
used in DNA replication of the lagging strand, uses ribonucleosides triphosphates to synthesis short RNA primers on the lagging strand, in eukaryotes these are about 10 nucelotides long and spaced about every 100 - 200 nucleotides.
DNA ligase
This enzyme seals a broken phosphodiester bond. DNA ligase uses a molecule of ATP to activate the 5′ end at the nick before forming the new bond . In this way, the energetically unfavorable nick-sealing reaction is driven by being coupled to the energetically favorable process of ATP hydrolysis.
DNA helicases
DNA helicases were first isolated as proteins that hydrolyze ATP when they are bound to single strands of DNA, the hydrolysis of ATP can change the shape of a protein molecule in a cyclical manner that allows the protein to perform mechanical work. DNA helicases use this principle to propel themselves rapidly along a DNA single strand. When they encounter a region of double helix, they continue to move along their strand, thereby prying apart the helix at rates of up to 1000 nucleotide pairs per second
Single-strand DNA-binding (SSB) proteins / helix-destabilizing proteins,
bind tightly and cooperatively to exposed single-stranded DNA strands without covering the bases, which therefore remain available for templating. These proteins are unable to open a long DNA helix directly, but they aid helicases by stabilizing the unwound, single-stranded conformation. their cooperative binding coats and straightens out the regions of single-stranded DNA on the lagging-strand template, thereby preventing the formation of the short hairpin helices that readily form in single-strand DNA. These hairpin helices can impede the DNA synthesis catalyzed by DNA polymerase.
replication fork
Y shaped region of a replicating DNA molecule at which the two daughter strands are formed and separate
regulated sliding clamp
whole ring slides freely along the DNA as the polymerase moves. The assembly requires ATP hydrolysis by the clamp loader, which hydrolyzes ATP as it loads the clamp on to a primer-template junction
On the leading-strand template, the moving DNA polymerase is tightly bound to the clamp on the lagging-strand template, each time the polymerase reaches the 5′ end of the preceding Okazaki fragment, the polymerase is released.
Telomerase
is a reverse transcriptase
with an iternal RNA template which extends telomeres in germ line cells, as DNAP cannot replicate the telomeres at the 3’ end of a eukaryotic linear chromsome
