Biochemistry - DNA Replication

Question 1

origin binding proteins

Answer 1

recognize origin sequences (A-T rich region) and separate the DNA strands locally, forming a replication bubble.

Question 2

origins of replication

Answer 2

A-T rich region,

Question 3

DNA helicase

Answer 3

Required to extend the movement of the fork. Catalyzes separation of dsDNA, moving from 5’ to 3’ direction. Requires ATP.

Question 4

SSB Proteins (SSBP)

Answer 4

Stabilizes the single-stranded molecules, preventing re-association of the two strands to protect the DNA from degradation. They bind cooperatively and are NOT enzymes.

Question 5

Key components for replication

Answer 5

dNTPs (deoxynucleotide triphosphates), Single-stranded template, a free 3’-OH group, RNA primer.

Question 6

Major proteins involved in Replication

Answer 6

Helicases, ssBP, primase, DNA polymerasea, Rnase H, DNA ligase, topoisomerases.

Question 7

Energy for incorporation of nucleotide

Answer 7

energy of the triphosphate. (NOT ATP hydrolysis)

Question 8

DNA polymerase (DNA pol)

Answer 8

Covalently attach nucleotides to the 3’-OH end of the growing daughter strand one at a time. add bases ONLY to existing chains, and can ONLY extend from 5’ to 3’ direction.

Question 9

Primase

Answer 9

Synthesizes short stretches of RNA that are complementary to the DNA template, called RNA primer.

Question 10

RNA primer

Answer 10

Provides a short, double stranded region consisting of RNA base-paired to the DNA template with a free 3’-OH group,the starting point for DNA polymerase.

Question 11

nucleoside reverse transcriptase inhibitor (NRTI)

Answer 11

Blocks viral DNA replication because it does not have a hydroxyl group on the 3’-carbon so when incorporated into viral DNA, no additional nucleotides can be added. Blocks HIV reverse transcriptase, an HIV enzyme, preventing HIV from replicating and lowers the amount of HIV in the blood.

Question 12

didanosine(ddI) and zidovudine (ZDV)

Answer 12

nucleoside reverse transcriptase inhibitor (NRTI) examples.

Question 13

Leading strand

Answer 13

Strand that is being synthesized continuously in the 5’à 3’ direction towards the replication fork.

Question 14

Lagging strand

Answer 14

Strand synthesized in the direction away from the replication fork as short pieces (“Okazaki fragments”) of DNA in the 5’ to 3’ direction. These discontinuous pieces are eventually joined together to form a continuous DNA strand.

Question 15

Polymerase delta

Answer 15

the major replicative enzyme

Question 16

RNase H

Answer 16

Recognizes RNA-DNA duplexes and removes the primer, leaving DNA portion intact. a target for drug development to block HIV genome replication.

Question 17

DNA ligase

Answer 17

joins Okazaki fragments together by forming a phosphordiester bond.

Question 18

Topoisomerases

Answer 18

Create nicks or cuts in DNA to relieve the torsional tension caused by twisting, then sealing the nicks. This allows the normal unwinding of DNA to occur during replication.

Question 19

Topoisomerase I

Answer 19

transiently cuts one strand of DNA.

Question 20

Topoisomerase II

Answer 20

Transiently cuts both strands of the DNA

Question 21

camptothecin

Answer 21

Topoisomerase I inhibitor, DNA supercoild won’t replicate quickly. Anti-Cancer.

Question 22

doxorubicin

Answer 22

Topoisomerase II inhibitor

Question 23

Topoimerase inhibitor mechanism of action

Answer 23

1) stably bind to DNA thus not generating a single strand DNA as a template for DNA replication. 2) prevent topoisomerases from re-sealing the DNA. Both will stop DNA replication.

Question 24

Telomeres

Answer 24

Ends of the linear chromosome composed of thousands of repetitive sequences of non-coding DNA that protect chromosome from damage. Single and double stranded.

Question 25

“end-replication problem"

Answer 25

If the newly-made DNA strands are not sufficiently replicated, successive rounds of DNA replication will produce daughter strands that are progressively shorter because there is no room to synthesize a new RNA primer at the extreme 3' end.

Question 26

telomerase

Answer 26

RNA-dependent DNA polymerase (ribonucleoprotein), uses its own RNA template to synthesize DNA, not the primer (see the next slide). Contains an RNA component (RNA template), and it uses the telomeric DNA sequences to extend the parental DNA.

Question 27

Basic Rules of DNA replication

Answer 27

i. Semi-conservative ii. Starts at the ‘origin’ iii. Bidirectional iv. Semi-discontinuous v. Synthesis always in the 5’to 3’ direction vi. RNA primers required