DNA replication Flashcards
What is the Evidence for semi-conservative replication?
o Meselson-Stahl experiment(1958)
o Bacteria grown in heavy 15N = DNA will contain this. (heavy band observed)
o Cells then transferred to 14N and grown for 2 generations.
o After two gen 2 bands are observed but after 1 only hybrid observed.
What is a replisome?
Wiki says: The replisome is a complex molecular machine that carries out replication of DNA. The replisome first unwinds double stranded DNA into two single strands. For each of the resulting single strands, a new complementary sequence of DNA is synthesized. The net result is formation of two new double stranded DNA sequences that are exact copies of the original double stranded DNA sequence.
What is a replisome made of?
o The most important part of which, when engaged with DNA, is DNA Polymerase.
What does DNA polymerase require to function?
a region of single stranded DNA.
Deoxynucleoside triphosphate precursors.
A 3’-OH group onto which the nucleotide is added.
What direction does DNA polymerase make DNA in?
o DNA Polymerase makes DNA in the 5’ to 3’ direction.
Describe DNA synthesis
• DNA synthesis via DNA Polymerase
o Incoming deoxynucleoside triphosphate pairs with the base on the template strand.
o Phosphodiester bond forms and pyrophosphate is released.
o DNA Polymerase synthesises only onto a 3’ end of a chain.
o Processive – attaches to replication fork and template the whole way through the reaction.
How is DNA synthesis initiated?
o DNA Polymerase cannot synthesise DNA from scratch (de novo).
o During initiation a small RNA primer (8-12 bases) is made by primase.
o DNA polymerase then starts the synthesis of the new DNA strands. The primer is ultimately replaced by DNA.
What is the origin of replication?
• Bacteria= DNA synthesis starts at a specific point on the chromosome – Ori (origin of replication).
o Local melting(splitting) of DNA at the Ori as well as assembly of the 2 replisomes.
o Replisomes the move away from Ori in opposite directions = bidirectional replication forks. (5’ – 3’ – in terms of new strand being built).
How is DNA replication terminated?
o Opposite side to Ori = termination region called ter.
o Two forks approach from opposite sides of ter.
• What is Leading and lagging strand synthesis
o During replication there is a leading (overall 5’-3’) & a lagging (overall 3’-5’) strand.
o The lagging strand is synthesised discontinuously in small segments called Okazaki fragments (made 5’-3’). These tend to be 1000-2000 bases.
o RNA primers tell DNA Polymerase where to start making the Okazaki fragments.
What does Ligase do?
• Replacing the primer using Ligase:
o RNA primer is removed on lagging strand by 5’-3’ exonuclease activity of DNA Polymerase.
o The DNA Polymerase then goes on to replace the missing bases.
o The ‘nicks’ between Okazaki fragments are joined by DNA ligase. (requires 1 ATP).
What protein factors are required for DNA replication?
o Initiator Protein = binds Ori & unwinds the DNA at the Ori. o Helicase = Unwinds DNA at the replication fork. o Topoisomerases (gyrase etc.) = relax DNA and stop overwinding of supercoiled DNA ahead of fork. o SSB (single stranded DNA binding protein) = protect DNA from damage, stop internal base pairing & stabilise lagging strand synthesis. o Primase = synthesis of RNA primer. o DNA Polymerase I = removes and replaces RNA primer. o DNA Polymerase III = synthesis of the leading and lagging strands. o DNA Ligase = joining Okazaki fragments.
What makes up the replisome?
o Primosome includes = Helicase & Primase. o DNA gyrase (topoisomersase). o SSB. o DNA Polymerase I&III. o DNA Ligase.
Describe the process fo proof reading and post replication repair.
o DNA polymerase is a template driven enzyme = a phosphodiester bond only forms if incoming nucleotide pairs with the template base.
o DNA polymerase proof reads = in an incorrect base is added DNA polymerase has a 3’-5’ exonuclease which removes the mismatched nucleotide.
o This results in an error rate of 1 in 107 bases
o E. coli = 1000 bp/s
o Post replication repair corrects proof reading errors
o This results in only 1 in 109-10 mistakes.
o 10,000-20,000 repaired a day.
What degrades DNA? How? What are the different types?
o Exonucleases cleave nucleotides at the end of a polynucleotide chain.
o Endonucleases cleave bonds within DNA chain e.g. DNase
o Restriction endonucleases recognise specific sequences & cleave the DNA.
