Chromosomal DNA Replication

Question 1

What 4 characteristics is nucleic acid synthesis governed by?

Answer 1

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

Question 2

DNA structure

Answer 2

It is double stranded and composed of 2 antiparallel strands. It has a 5’ phosphate and a 3’ hydroxyl group.

Question 3

Watson-Crick Base Pairing

Answer 3

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.

Question 4

DNA template is READ _________ and MADE ________

Answer 4

3’ –> 5’; 5’ –> 3’

Question 5

In order for replication to occur, 3 things are needed:

Answer 5

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

Question 6

Assess the unique properties of DNA Polymerases

Answer 6

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’

Question 7

Describe Okazaki Fragments and their function

Answer 7

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.

Question 8

What is meant by DNA replication being bidirectional?

Answer 8

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.

Question 9

DNA synthesis utilizes what 6 specialised mechanisms?

Answer 9

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

Question 10

Initiation: Describe important features of the replication origin and the mechanism for the fork initiation reaction.

Answer 10

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.

Question 11

Primase

Answer 11

The enzyme that makes and places the RNA primers

Question 12

Initiator Proteins

Answer 12

Proteins that recruit helicase to unzip DNA. They also start to destabilize the helix

Question 13

Helicase

Answer 13

It is an allosteric motor protein that unwinds the DNA using a lot of ATP

Question 14

Single-Strand Binding Proteins

Answer 14

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.

Question 15

Explain the roles of primers and why they are RNA and not DNA; compare the synthesis and removal of primers.

Answer 15

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.

Question 16

Explain the loading of DNA polymerase and what is involved.

Answer 16

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.

Question 17

RNA Primer

Answer 17

Synthesised by primase, removed by RNase nuclease.

Question 18

Ligase

Answer 18

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)

Question 19

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.

Answer 19

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.

Question 20

Topoisomerase

Answer 20

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

Question 21

Topoisomerase I

Answer 21

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.

Question 22

Topoisomerase II

Answer 22

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.

Question 23

In what 3 ways does Eukaryotic Replication differ from prokaryotic replication?

Answer 23

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.

Question 24

DNA Replication occurs _____ during the cell cycle and during ___ phase

Answer 24

once; S-phase

Question 25

How many origins of replication do eukaryotic chromosomes have?

Answer 25

There are many of origins of replication and they occur in “groups”. For instance, in one S-phase one group of origins would be used but in another cell cycle the others are used, etc.

Question 26

Describe Eukaryotic control of replication

Answer 26

During the G1 gap phase, just before S-phase, the origin recognition complex (ORC) binds to the origin of replication. Then, the ORC proteins recruit helicase, becoming the pre replicative complex. This complex sits there until a kinase is activated in S-phase which then phosphorylates both helicase and the ORC proteins which activated helicase to start unwinding the DNA. This also recruits the polymerase. The ORCs, by being phosphorylated, are “marked” as starting replication and until this phosphorylation signal is removed, replication will NOT occur again.

IT ENSURES THAT EACH REGION OF DNA IS REPLICATED ONLY ONCE PER CELL CYCLE

Question 27

Identify important chromosomal DNA sequences and their functions.

Answer 27

There is centromeric DNA. It is more highly condensed and binds the kinetochore for anaphase.

Telomere sequences: they are G-rich regions that prevent the ends of chromosomes from recombination, fusion, and being recognized as damaged DNA and degraded. They form a loop.

Origin of Replication: and A-T-rich region of the genome. It is where replication begins.

Question 28

Compare early and late replicating DNA

Answer 28

Early replicating DNA is likely to be euchromatin or coding regions as it is less tightly compacted. Late replicating DNA is likely to be heterochromatin as it is more highly condensed. However, it is important to note that ALL DNA is replicating during S-phase, heterochromatin and euchromatin alike.

Question 29

Describe the fate of nucleosomes during DNA replication

Answer 29

During the DNA replication, the histones and DNA must be dissociated from one another. The parental chromatin will mark the histone tails and variants and these histones are then reused on the two daughter strands. This is how epigenetic information is passed on. It involves histone chaperone proteins that work together to remove and reload histones on newly synthesized DNA. Because there is double the DNA only half of the parental histones with modifications will be present with half newly synthesized histone proteins. Reader and writers will then come in and read the modifications and then write these modifications on the adjacent histones that do not have the modification, now giving it to them. The readers and writers are crucial in remodifying the nucleosomes.

Question 30

Telomerase

Answer 30

It is a riboprotein meaning it has an RNA template unit to synthesize the G-rich telomere region and a protein unit. Not all cells express telomerase. It is primarily expressed in germ cells and stem cells and cancer cells.

Question 31

Explain the “end-replication” problem

Answer 31

Once an RNA primer is removed, there is a chunk of DNA that has not been replicated left on the lagging strand. Over time, if this is not fixed, the lagging strand ends will continue to shorten, eventually degrading non-telomeric regions and thus damaging coding DNA. By using telomerase, the telomeres can be extended on the opposite strand, allowing room for another primer to bind and thus DNA polymerase to extend and replicate the telomere regions as well.

Question 32

Describe the mechanism for the addition of telomere repeat sequences.

Answer 32

Telomerase contains an RNA template region that codes for the G-rich telomere region. The telomerase extends the 3’-overhang of the parental strand in order to provide more room for a primer to bind to it and make a final okazaki fragment via polymerase.

Question 33

Analyze the relationship of telomere length with cellular lifespan.

Answer 33

The shorter the telomeres, the older the person likely is.

Question 34

Explore the potential of a telomerase template antagonist as an anticancer agent

Answer 34

By building a sequence to antagonistically bind to the telomerase, it would knock out the function of the telomerase. This would result in the cell not extending the telomeres, eventually leading to senescense and thus cell death.

Question 35

Identify common DNA-repeat expansion diseases

Answer 35

Myotonic Dystrophy SCA, spinocerebellar ataxia; DRPLA, denatorubral pallidoluysian atrophy; SBMA spinal and bulbar muscular atrophy. Also Huntington’s disease.

Question 36

Describe significant features of DNA-repeat expansion diseases

Answer 36

These diseases show anticipation. In other words, they are increasing in severity over time. Furthermore, they are all a result of addition of repeated sequences. Above a certain number of repeats, you would no longer have appropriate function, splicing, or phenotype. Through the generations, you tend to see more and more repeats as well. Slipped mispairing during DNA replication is likely to be the cause. DNA polymerase gets confused while it is replicating repetitive sequences and slips off and in repetitive sequences, it doesn’t really know where it was. Can get looping and then expansion in the next cycle.

Question 37

Relate location of the DNA expansion with effect on transcription and translation.

Answer 37

In promoter: no transcription
In 5' UTR: no transcription
In intron: no transcription
In ORF: Get transcription and translation
In 3' UTR: ????? unknown

Question 38

Relate genetic features with clinical features of a DNA repeat expansion disease

Answer 38

One would expect that as there are more of the repeats genetically, that clinically the disease would be more severe.