Chromosomal DNA Replication Flashcards
(38 cards)
What 4 characteristics is nucleic acid synthesis governed by?
1) A pre-existing nucleic acid strand is copied by rules of watson-crick base pairing
2) Nucleic acid strands grow in only one direction: 5’ –> 3’
3) Polymerases synthesize nucleic acids
4) Duplex DNA synthesis requires a special growing fork because the strands are antiparallel
DNA structure
It is double stranded and composed of 2 antiparallel strands. It has a 5’ phosphate and a 3’ hydroxyl group.
Watson-Crick Base Pairing
A to T; G to C; This is the mechanism by which you can get fidelity during replication. C ONLY base pairs with G and A ONLY base pairs with T. If this is wrong, then there is a mutation and repair/proofreading mechanisms can notice this.
DNA template is READ _________ and MADE ________
3’ –> 5’; 5’ –> 3’
In order for replication to occur, 3 things are needed:
1) Accurate base pairing
2) An RNA primer to start the process
3) Have to have a 3’ hydroxyl group available to react with the triphosphate of the incoming nucleotide to form the phosphodiester backbone. The hydroxyl group performs a nucleophilic attack on the first phosphate group to give off pyrophosphate which will later be converted to 2 phosphates
Assess the unique properties of DNA Polymerases
DNA polymerase requires a 3’-hydroxyl group in order to add the next nucleotide. The incoming DNTP (deoxyribonucleotide triphosphate) is incorporated at the 3’-OH. The Polymerase also requires watson-crick base pairing and a primer. Everything has to be aligned appropriately in the catalytic site of the polymerase in order for replication to occur correctly. It requires the RNA primer, a 3’-OH, and enough space for the DNTP to enter. Once it has the correct configuration, there is a conformational change in polymerase, locking everything into place. It then loosens a little, inputs the next base, and continues. Polymerase also ONLY READS 3’ –> 5’ , synthesizing the new strand 5’ –> 3’
Describe Okazaki Fragments and their function
Because DNA Polymerase can only read the template stand 3’ –> 5’, DNA replication cannot occur in both directions. Therefore, the leading strand occurs continuously, but, in order to get complete synthesis, there are lagging strands or okazaki fragments. They are also made 5’ –> 3’ however they are made from the replication fork toward the origin of replication. They also require multiple RNA primers (one per fragment) and thus are not a continuous strand.
What is meant by DNA replication being bidirectional?
It means that a primer is added to each parent strand, using them both as templates. However, since DNA is antiparallel, the polymerase synthesizes the two leading strands 5’ –> 3’, but this is in opposite directions. Therefore, you get the synthesis of a strand in one direction, and the synthesis of the other strand in the opposite direction, being bidirectional. There are two replication forks and the leading strand continues to be sythesized toward them as they keep opening.
DNA synthesis utilizes what 6 specialised mechanisms?
1) Initiation: have to start it somewhere
2) Unwinding: have to unwind the stable configuration of DNA
3) Priming: Need an RNA primer for polymerase to start at
4) Unidirectional fork movement: one replication fork moves in one direction, the other in another.
5) Untangling: Topoisomerase untangles the DNA
6) Termination: Have to stop
Initiation: Describe important features of the replication origin and the mechanism for the fork initiation reaction.
Initiation begins at the origin of replication. The origins tend to be 1) A-T-rich because they are base pairs that are easier to open 2) specific initiator proteins bind to the origin to start replication. 3) helicase motor proteins are then loaded on to each strand to unzip the DNA 4) Primase synthesizes an RNA primer which allows DNA polymerase to start replication. The primers then must be added for the lagging strand too. It occurs after the leading strand, thus it is lagging.
Primase
The enzyme that makes and places the RNA primers
Initiator Proteins
Proteins that recruit helicase to unzip DNA. They also start to destabilize the helix
Helicase
It is an allosteric motor protein that unwinds the DNA using a lot of ATP
Single-Strand Binding Proteins
SSBs prevent reannealing of the two template strands without preventing base pairing between the newly synthesized strand and the parent strand. They bind to the single strand of DNA to keep it single stranded and also prevent intra-strand h-bonding. They bind cooperatively, once one binds, the others bind more easily. They bind in such a way to the DNA in which they straighten out the DNA and the phosphodiester bonds but still leave the DNA bases exposed so the polymerase can read them even wit the SSBs there. They are the most crucial on the lagging strand.
Explain the roles of primers and why they are RNA and not DNA; compare the synthesis and removal of primers.
DNA synthesis starts with RNA because it needs to be able to put to bits of sequence together efficiently as well as being able to distinguish a region that was copied as potentially having errors that you want to fix. Because primase can put two bases together without a template very easily, most bases may match the template correctly but others may be mistakes. Therefore, this region has to be easily identified as one that needs to be re-looked at. The primer is synthesized by primase and removed by RNase nuclease.
Explain the loading of DNA polymerase and what is involved.
A regulated sliding clamp permits protective DNA synthesis on the leading strand and rapid reassembly of the replication complex on the lagging strand. A clamp loader protein, ATP, and the sliding clamp protein associate causing the sliding clamp to open. Then, the clamp loader brings the complex to DNA, loading the sliding clamp on the DNA. ATP dissociates causing the sliding clamp to close around then DNA and the clamp loader to dissociate. DNA Polymerase then binds to the sliding clamp.
RNA Primer
Synthesised by primase, removed by RNase nuclease.
Ligase
Forms/reforms the phosphodiester bonds in the DNA backbone. it seals the nicks using ATP. Ligase, using ATP, will add a phosphate to the monophosphate nucleotide. Then, the 3’-OH can react with the diphosphate (specifically with the first phosphate) to form a phosphodiester bond. Then get the release of the outer phosphate (AMP)
Understand that DNA polymerase has editing as well as polymerizing function, why this is important for high fidelity replication, and potential impact of mutations in the proofreading domain of DNA polymerase.
DNA “proofreads” as it is synthesizing DNA. If it incorporated the wrong base, it has 3’ –> 5’ exonuclease activity in which it will chew back and replace the base with the correct one and then continue replication. There is an allosteric change in the polymerase when the wrong base is added such that it moves it to the editing site to be replaced. This is important for fidelity because it now has less errors in replication! If it didn’t have this function there would be many more mutations in the genome. If there were mutations in the proofreading domain of the DNA polymerase it would lose the editing ability and the DNA would be more prone to mutation. If the mutations were to occur in the genes, it could be detrimental causing colorectal adenomas and carcinomas.
Topoisomerase
There is a winding problem that arises during DNA replication. Because DNA is so long it acts as if it were anchored, not being able to move and unwind easily therefore building up torsional stress (about 10bp per turn). There are two topoisomerase enzymes, topoisomerase I and II
Topoisomerase I
It breaks 1 strand. It has a tyrosine at the active site that will attack the phosphodiester bond, binding to the phosphate group. This will allow a DNA double helix to unwind around the other strand. The bond then reforms.
Topoisomerase II
It is breaking 2 strands. When 2 DNA double helices are interlocked, it comes in and cleaves one of the strands, hold it in place, bringing it around the other strand, and then reforms the helix. These are targeting in cancer treatment to form double stranded breaks in the DNA, however, a side effect is when apoptosis doesn’t occur you just introduced more cancer.
In what 3 ways does Eukaryotic Replication differ from prokaryotic replication?
1) It is compartmentalized within the nucleus, partitioning it from the site of synthesis of replication proteins and precursors and from extracellular stimuli that may trigger initiation of synthesis
2) It begins at multiple origins, activated throughout S phase in a precise and temporally regulated manner
3) The nucleosomal proteins must be duplicated along with the DNA to maintain proper chromosomal organization.
DNA Replication occurs _____ during the cell cycle and during ___ phase
once; S-phase
