Biochemistry - Lectures 11

Front 1

DNA Replication

Back 1

Takes about 40 minutes in bacteria, in eukaryotes it can vary from 1.4 to 24 hours.

Front 2

Components needed to build DNA

Back 2

1) Building block - deoxynucleoside triphosphate
2) Piece of DNA as template
3) DNA polymerase
4) Need magnesium ions
5) NEED primer, DNA polymerase requires 3' - OH to build from

Front 3

DNA Polymersase

Back 3

Template strands runs from 3' to 5'. Synthesis new strand 5' to 3' but reads 3' to 5'. Brings in deoxyribonucleoside and then cleaves phosphate to allow for attachment to 3'-OH to ribose sugar above. Cleaves pyrophosphate off energy source.

Front 4

Acyclovir Triphosphate

Back 4

Is a guanosine analog which is first phosphorylated by viral thymidine kinase to acyclovir triphosphate. Incorporates itself into viral DNA and because it lacks 3' - OH group causing termination of DNA synthesis.
Inhibitor of viral DNA polymerase - Herpes Virus

Front 5

Cytosine Arabinoside and Adenosine arabinoside (Cidarabne)

Back 5

Both have -OH group but planar configuration is not normal and results in kinky phosphodiester bond and induces DNA damage and inhbits activity of DNA polymerase. These are anti-cancer drugs.

Front 6

DNA Exonuclease in Tautomeric shift

Back 6

DNA polymerase is tricked where bases undergo shift where it mimics another base results in the wrong complimentary base by DNA polymerase. Shifts are unstable and will shift back to normal base state and will disengage with base.
DNA polymerase contains 3' to 5' proofreading exonuclease, which chews back, repairs the mutation and than continues.

Front 7

Bacterial Genomes

Back 7

Are circular and have a single ORI. Undergoes theta replication. Starts at one point along circle. DNA synthesis begins both ways until 2 full circles are created

Front 8

Rolling-Circle Replication

Back 8

Occurs in bacteriorphage and plasmids. Starts at ORI on goes in one direction on the circle and the circle is cut and the strand is removed as new strand is synthesis.

Front 9

Linear Replication

Back 9

Occurs in eukaryotes. Have multiple point of replication and occurs bi-directinally.
Human genome contains 3.4 billion base pairs and rate of replication is 2000bp/minute.

Front 10

Leading strand

Back 10

Only requires one primer and then has continues synthesis towards replication fork as it opens the strand.

Front 11

Lagging strand

Back 11

Discontinuous synthesis, replication fork is behind the lagging strand. Must wait for strands to open up before new RNA primer can be added. Called ocazaki fragments.

Front 12

Initiator proteins (DNAa protein)

Back 12

Proteins that bind to ORI (to certain sequences). They initially break the hydrogen bonds between bases.

Front 13

Helicase Inhibitor (DNA -C)

Back 13

Deliverse helicase to the ORI and the replication fork. Once it delivers, it disengages and helicase becomes active.

Front 14

DNA Helicase (DNA-B)

Back 14

Continues the process of unwinding the the helix and bins to primase to form primosone.

Front 15

DNA Primase

Back 15

An RNA polymerase that synthesis a RNA primer on the leading and lagging strands to enable DNA polymerase to synthesize. Provides

Front 16

DNA Polymerase III

Back 16

Synthesizes the leading and lagging strands from RNA primer. Has 5' to 3' polymerase activity and has 3' to 5' exonuclease activity (proofreading activity)

Front 17

DNA Polymerase I (RNAseh and FEN-1 in eukaryotes)

Back 17

Responsible after replication has occurred to remove RNA primer and replace with DNA.
1) 5' to 3' polymerase activity
2) 3' to 5' exonuclease activity (when replacing primer)
3) 5' to 3' exonuclease activity (when removing primer)

Front 18

Single Stranded Binding Protein (SSB)

Back 18

Bind to single stranded DNA during replication to prevent it from reannealing. Coats the single strand and comes off after DNA polymerase has passed. SSB prevents deletion mutation of stand folding.

Front 19

OriC

Back 19

Contains DNA sequences that are A-T rich. Contains this because A-T base strands have only 2 hydrogen bonds compared to G-C, making it ideal location to for start replication synthesis.

Front 20

Initiation of Replication

Back 20

1) Initiator proteins bind to ORI sequence
2) Breaks hydrogen binding and separation occurs
3) Helicase inhibitor protein delivers helicase to template
4) Helicase clamps around each ssDNA
5) Helicase proceeds to unwind bi-directionally
6) DNA Primase comes in and drops RNA primer
7) DNA polymerase comes in and binds to RNA primer

Front 21

How are lagging stands joined?

Back 21

By DNA Ligase which uses ATP to bind Phosphate of primer replaced end to next fragment.

Front 22

How does DNA polymerase III know when to stop synthesizing in the lagging strand when it hits previous fragment?

Back 22

DNA Pol III has low affinity to DNA. Clamp protein holds DNA pol onto template. When clamp hits dsDNA, it disengages and DNA Pol II also falls off.

Front 23

As helicase unwinds DNA, it is creating positive supercoiling ahead of replication form and this is alleviated by?

Back 23

By Topiosmerase 1 (DNA gyrase). Which forms covalent bond with DNA and breaking phosphodiester bond (nicks it) which then allows relieves tension.
Topo 2 can also use ATP to introduced negative supercoiling to counteract.

Front 24

Ciprofloxacin

Back 24

Is a quinolone drug that inhibits bacterial DNA gyrase. Used for respiratory and urinary tract infections and used to treat anthrax. Inactivating gyrase, causing positive supercoiling tension to build up and stopping DNA synthesis.

Front 25

Telomeres (eukaryotes)

Back 25

Because its linear, removing the RNA primer results in a GAP (telomeres). Only way to fill gap is if there is a free -OH group. Not fixing results in shorter chromosomes (old age). Telomeres are repeated sequences (T-G rich region)

Front 26

Telomerase

Back 26

Similar to reverse transcriptase enzyme. Which uses RNA as a template to synthesis DNA. Enzyme holds within it a short RNA, which is complimentary to telomere sequences and binds to overhang. Reverse transcriptase will extend the overhang, repeats to extend overhang.
New DNA synthesized is extra (non-encoding). This overhand allows primase to come in and add primer so gap can be synthesized.
Cancer cells have high telomerase activity.

Front 27

Differences in eukaryotes

Back 27

1) Have multiple ORI
2) RNA primer removed by RNAaseH and FEN!
3) Different DNA polymerase
beta + gamma - DNA repair

Front 28

Polymerase - alpha

Back 28

-synthesize RNA primer on leading and lagging strand
-contains primates activity and initiates DNA syntehss
-no exonuclease

Front 29

Polymerase - delta

Back 29

-Synthesizes DNA from leading and lagging strands
-Main replicative enzyme
-3' to 5' exonuclease in proofreading

Front 30

Camptothecin

Back 30

Eukaryotic - an antiancer drug which inhibits topo 1 resulting in DNA breakage

Front 31

Etoposode

Back 31

Eukaryotic - another anticancer drug that inhibits activity of topo 2

Front 32

Antinomycin D

Back 32

A inhibitor of replication in both eukaryotic and prokaryotic. It is an intercelator which inserts itself in the double helix and stabilizes it. Does not allow for unwinding, halts transcription.