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1

What is Camptothecin?

it is isolated from the happy tree, it stops topoisomerase 1
from working preventing DNA replication. it has anti cancer activity but many side effects.

2

What is Topoisomerase 1

Its the enzyme responsible for DNA replication. it removes the supercoils that arise from DNA unwinding.
Induces a DNA break to relieve the supercoiling, then reseals it. Camptothecin stops the resealing and pervents topoisomearase from working.

3

Define Conservative replication

First replication, yeilds 1:1 old and new strand. 2nd replication yeilds 1:3 old and new strands

4

Define Dispersive Replication

Bits get replicatied and glued together,all DNA yeilds a mixture of old and new

5

Define Semi Conservative

Produces new strands, but also produces mixture DNA with new and old strands glued together.

6

Define the Meselson and Stahl experiment.

Used equilibrium density gradient centrifugation to distinguish between old and new DNA.
they band DNA with 15n and 14n new DNA is lighter than old DNA so when centrifuged will separate into old and new strands of DNA. They centrifuged the DNA 4 times.
1st with 15n whichs marks the dna with a heavy band.
2nd they let the dna replicate with addition of 14n, they spin again.
3rd spin they get new and old DNA split. The new is marked with 14n and the old is marked with 15n.
This proves that, all DNA molecules are a mixture of old and new DNA. and that new DNA is made from old DNA, and proves the Semi Conservative model.

7

List the basic replication requirements

* DNA template
* Raw materials, building blocks
*Enzymes and other proteins that read the template and assemble the new strand.

8

Template?

An original DNA strand to replicate from.

9

Replication fork ?

replication goes in two directions, produces two new strands at the same time.

10

Leading strand?

A strand in replication that replicates continuously.

11

Lagging Strand?

A stand in replication that replicates discontinuously, or in sections.

12

What are the fragments produced in the lagging strand called?

Okazaki Fragments

13

list the 4 Mechanism of Replication in eukaryotes ?

* initiation
* Unwinding
*elongation
* Termination

14

what is proof reading?

If a wrong nucleotide is found it will be cut out and replaced with the correct base. occurs during replication

15

What is mis-match repair?

After replication is completed, if any mis matched nucleotides are found they are cut out and replaced (occurs after replication)

16

Explain the role of DNA polymerase 1 ,111 ?

DNA polymerase 1 is the enzyme that adds nucleotides to a free 3'-OH group and removes RNA primers.
DNA polymerase 111 elongates a new nucleotide strande from the 3'-OH group provided by the primer

17

DNA Ligase?

This enzyme will seal nicks in the DNA sugar phosphate backbone at the end of replication.

18

DNA Primase?

Produces short RNA prmers with free 3'-OH ends.

19

DNA helicase?

Unwinds DNA at replication fork

20

Initiator Protein?

Binds to origin and separates Strands of DNA to initiate replication.

21

DNA Gyrase?

Moves ahead of the replication fork, making and resealing breaks in the double helical DNA to release torque that builds up as a result of unwinding at the replication fork.

22

Single-strand-binding proteins?

Attach to a single stranded DNA and prevent reannealing

23

DNA Initiation

*Initiation starts at the origin of replication (oriC)
*Initiator proteins bind to oriC and cause unwinding of a short section of DNA.
*DNA helicase and other single-strand-binding proteins will subsequently bind.

24

What do SSB Proteins do ?

Keep the strands separated.

25

Bidirectional?

DNA replication occuring Anti-parallel to each other.

26

Eukaryote Replication

Is more complex, contains many origins of replication that all have to fire at once and once only.
is separated in two steps
- origins are licensed by replication licensing factor.
- replication is initiated.
Telomeres play an important role due to linear DNA.
nucleosomes form new and old histone proteins.

27

Differences between circular and linear replication.

circular only has one origin of replication, and occurs completely around adding nucleotides to the free 3'-OH group.

Linear has multiple origins of replication, happens bidirectional in a anti parallel order, consists of a leading strand and a lagging strand. needs primers to be synthesised, elongation of DNA strand and the removal of those primers.

28

What happens to the telomeres during replication?

Telomeres are the ends of eukaryotic chromosome. they are replicated by an RNA- protein complex called telomerase. which adds nucleotides to the GC rich strand of telomere. The shortening of Telomeres may contribute to ageing.
mice deficient in telomerease show premature ageing. Somatic cells can be made that have active telomerase, which will divide indefinitely.

Werner Syndrome, autosomal recessive caused by a defect in WRN.

29

Dyskeratosis?

The X-linked form causes a defect in dyskerin which processes the RNA component of telomerase

Telomeres in the germ cells are shortened and each successive generation has shorter telomeres and more severe disease. ( anticipation)

* cancer cells often express telomerase

30

Transcription?

Is the synthesis of RNA molecules using DNA as a Template.

31

The Primary and Secondary structure of Transcription.

Primary = linear
Secondary= Folded

32

Define mRNA, tRNA, rRNA.

mRNA= Messenger RNA, found in both nucleus and cytoplasm. carries the genetic code for protein.
tRNA= Transfer RNA found in the cytoplasm. Helps incorporate amino acids into a polypeptide chain.
rRNA= Ribosomal RNA, in the cytoplasm. Structural and functional components of the Ribosome.

33

Template Strand

A single strand of DNA that codes for a single strand of RNA.

34

Describe prokayotic transcription

Bacterial Transcription consists of 3 stages:
1. initiation.
2. elongation.
3. termination.
* initiation is the main step for control, it involves a promoter recognition, formation of transcription bubble, creation of first complementary base pairings, escape of machinery from promoter.
Its promoter consists of :
- TATA box at -10
- -35 consensus sequence
- sometimes a third upstream element
* RNA polymerase is positioned at start site (+1)

35

Termination in prokaryotes

Termination is either Rho dependent or Rho independent.
inverted repeats repeats are transcribed and form a hairpin loop in Rho independent termination.
In bacteria polycistronic mRNA can be formed multiple genes with one promoter and one terminator.

36

Transcription in Eukaryote cells

No generic promoters found, depends on whether it is recognised by RNA ploymerase i,ii,iii
many proteins take part in promoter binding and RNA polymerase cannot start transcription by itself.
DNA structure has to be modified as well.
Termination is NOT important.
Transcription in Archea is NOT important.

37

Transcription Initiation in Eukaryote cells

Transcription Initiation in eukaryote cells needs ;
- general transcription factors ( forms the basal transcription apparatus )
- transcription activator proteins.
-promoter sequences
-enhancer sequences.