Lecture 2: DNA Replication - The Process (Completed up to Slide 12) Flashcards
Completed up and including Slide 12
Why is it not possible for one of the new strands to be replicated 5’ to 3’ and the other 3’ to 5’?
There are no DNA polymerases which can synthesise DNA in the direction of 3’ to 5’.
What are the different strands called during DNA replication and how are they different?
The leading strand and the lagging strand. Each replication fork will have a leading and a lagging strand. The leading strand is continuously synthesised 5’ to 3’, in the direction of the replication fork movement. The lagging strand is also synthesised 5’-3’ but discontinuously, as a series of short DNA pieces called Okazaki fragments. These are then joined to make long nascent DNA chains. Okazaki fragments were discovered when E. coli was pulse labelled with [3H]thymidine. After transferral to unlabelled medium the size of the labelled fragments increased over time. Elongation of the lagging strand is in the opposite direction to the direction of replication fork advance. While leading strand synthesis requires a single RNA primer, lagging strand synthesis requires multiple RNA primers, one for each segment – synthesised by DNA primase. DNA replication is therefore semi-discontinuous.
How is DNA replication terminated?
Replication terminates at specific Ter sites. At the end of replication, the two double-stranded daughter chromosomes are interlocked and need to be separated by a type II DNA topoisomerases.
Discuss the origin of replication in prokaryotes.
From a single origin (OriC, AT-rich in E.coli) the 2 replication forks advance in opposite directions at constant speed & meet up about halfway round chromosome. OriC is bound by an initiator protein DnaA. Opens up a ~45 bp segment into single strands. DnaC binds and permits helicase, DnaB, binding.
In prokaryotes, what does a) DnaA, b) DnaB and c) DnaC do?
a) DnaA binds to OriC (the origin of replication in prokaryotes) and opens up a roughly 45 bp segment into single strands. b) DnaB is a helicase, which pries apart the DNA duplex ad the replication fork moves. c) DnaC binds to OriC and allows DnaB to bind
Discuss the origin of replication in eukaryotes.
There are thousands of origins and replication forks in a eukaryotic cell, one every 3 to 300 kb. A eukaryotic chromosome typically contains 60 times more DNA than a prokaryotic chromosome. If operating from a single origin replication could take months. Yeast has ‘Autonomously Replicating Sequences’ (ARS, 11 bp).
Which 102 kDa enzyme discovered in 1957 by A.Kornberg was originally thought to be responsible for all of DNA replication?
DNA Polymerase I (the I was added later when other DNA polymerases were discovered).
What are the activities of DNA Polymerase I?
5’ – 3’ polymerase activity 3’ – 5’ exonuclease activity - very important for proof-reading 5’– 3’ exonuclease activity - allows degradation of a strand ahead of the advancing polymerase. - Useful in DNA repair and removal of RNA primers (‘Nick Translation’ on the lagging strand)
How are the activities of DNA Polymerase I localised in its structure?
If DNA Polymerase I is enzymaticallly cleaved by protease subtilisin, two fragments result. 68kDa = Klenow fragment 3’-5’ exonuclease + polymerase 35kDa = 5’-3’ exonuclease The Klenow Fragment is often used in labs when you require DNA synthesis but no destruction of strand. It is often used for end “filling”. DNA polymerase I (‘Nick translation’) can be used for the radioactive labelling of single-stranded DNA probes. Use DNase to create single stranded nicks in double stranded DNA. Add Pol I with 32P-labelled dNTPs. It replaces unlabelled nucleotides with labelled nucleotides.
What happened in 1969 which challenged the current idea of DNA replication at the time?
In 1969, Paula Delucia & John Cairns Cairns found a Pol I mutant, a bacterium without a functional DNA Polymerase I. It was strong & viable, so they concluded that DNA Polymerase I we not as critical as was previously thought.
What happened in 1971 which confimred that DNA Polymerase I is not solely responsible for DNA replication?
In 1971 Thomas Kornberg & Malcolm Gefter discovered 2 new polymerases (II & III).
Compare DNA Polymerase I, II and III.
DNA polymerase II is thought to be a backup polyermase in prokaryotic DNA replication. It has the 3’ to 5’ exonuclease activity so can proof read, but does not have the 5’ to 3’ exonuclease activity. It is the smallest of the three DNA polymerases and has an intermediate number of subunits, polymerisation rate and processivity.
Processivitiy is the number of nucleotides added before the enzyme dissociates.
DNA polymerase I is smaller than DNA polymerase III and does not have any subunits, whereas DNA polymerase III has 10 or more.
DNA polymerase III has a much higher polymerisation rate and processivity than DNA polymerase I.
Both DNA polymerase I and DNA polymerase III have 5’ to 3’ polymerase and 3’ to 5’ exonuclease activity, but only DNA polymerase I has 5’ to 3’ exonuclease acitvity.
