Dna Replication

Front 1

DNA replication proceeds from an origin

Back 1

i. Eukaryotic chromosomes have 1000-2000 separate origins of replication per chromosome
ii. Bacteria have single chromosomes and possess a unique replication origin

Front 2

Bidirectional replication in DNA

Back 2

1. Two replication forks proceed in opposite directions from origin
2. Must be unraveled
3. 5’→3’
4. Initiated from pre-existing primers→ short section of RNA that is complementary to the template strand

Front 3

Leading strand

Back 3

a. Replication is continuous→ DNA is synthesized uninterruptedly from a single RNA primer

Front 4

Lagging strand

Back 4

a. Replication is discontinuous→ proceeds from multiple primers and results in several short DNA sequences to be joined later

Front 5

Topological problems with DNA replication

Back 5

a. DNA molecules not linear→ “closed loop”
b. Supercoiling must be avoided

Front 6

DNA polymerase

Back 6

i. (dNMP)n+ dNTP → (dNMP) n+1 + PPi → 2 Pi
ii. Only deoxyribonucleotide triphosphates can serve as substrates
iii. Requires a free 3’-OH terminal from which to start

Front 7

RNA polymerase

Back 7

i. Necessary for synthesis of RNA primers for DNA replication
ii. Adds oligonucleotide of 10-60 bases to 3’-OH
iii. Acts on both leading and lagging strands
iv. “Primase” in E. coli

Front 8

Okazaki fragments

Back 8

i. Short sections of primer RNA plus DNA that form on lagging strand

Front 9

Helicase

Back 9

i. Carry out unwinding
ii. Bind to single-stranded DNA
iii. Require ATP
iv. E. coli→ rep protein and dnaB+dnaC→ form part of replisome

Front 10

Replisome

Back 10

i. Total proteins and enzymes required for DNA synthesis at replication fork

Front 11

Supercoiling

Back 11

i. Supercoils introduced into DNA when a closed circular duplex is twister around a central axis

Front 12

Topoisomerase

Back 12

i. Relieve tension ahead of replication fork that is introduced via unwinding of DNA strands (positive supercoiling)

Front 13

Type I→ DNA topoisomerase

Back 13

1. Makes a break or nick in one strand of DNA helix
2. Passes other strand through break to relax supercoil
3. Does not require ATP

Front 14

Type II topoisomerase

Back 14

1. Produce an enzyme-bridged break in both strands of DNA
2. Another region of duplex DNA is passed through the gap by the enzyme
3. Two supercoils are removed in one step
4. Requires ATP

Front 15

Target for drugs-- topoisomerase

Back 15

5. Target molecule fro some of the most important anti-cancer drugs→ Adriamycin and etopside
a. Ciprofloxacin→ antibiotic active against gyrase

Front 16

Single-strand binding proteins (SSB)

Back 16

1. Keep the separated strands as single strands
2. Affinity for single-stranded DNA 1000x greater than for double-stranded DNA

Front 17

DNA polymerase III

Back 17

1. Enzyme responsible for extending growing DNA strand to 5’ ribonucleotide of primer→ DNA p. I takes over
2. Processive→ never dissociates until entire chromosome has been replicated
3. Two at each replication fork→ 4 in replication bubble

Front 18

DNA polymerase I

Back 18

1. Possesses 5’-3’ exonuclease activity → cuts out RNA primer one nucleotide at a time
2. Simultaneously fills in corresponding dNTP to DNA template
3. 3’-5’ exonuclease activity → editing or proofreading

Front 19

DNA ligase

Back 19

1. Joins Okazaki fragments

Front 20

DNA replication at oriC in E. coli

Back 20

a. dnaA proteins bind to oriC to initiate replication → separates both strands in this region
b. dnaB+dnaC complex (dnaB is a helicase) bind to separated strands at oriC to form replisome
c. Primase joins the complex to begin synthesis of RNA primer

Front 21

DNA polymerase alpha

Back 21

i. Responsible for formation and extension of RNA primers

Front 22

DNA polymerase beta

Back 22

i. Involved in DNA repair in the nucleus

Front 23

DNA polymerase gamma

Back 23

i. Localized in mitochondria for replication of mitochondrial DNA

Front 24

DNA polymerase delta

Back 24

i. Synthesizes DNA on leading strand

Front 25

Dissociation of histones

Back 25

i. Association of the nucleosome and DNA at site of DNA synthesis is weakened through acetylation and phosphorylation of histones
ii. Makes histones more negative

Front 26

Multiple initiation sites

Back 26

i. E. coli replicates 100,000 base pairs per minute
ii. Eukaryotes→ 500-5000 base pairs/min.
iii. Inequity over come by various eukaryotic initiation sites

Front 27

ORC

Back 27

i. Origin recognition complex
ii. Must bind at origins of replication sequences
iii. Licensing factor→ must be bound near each origin to ensure the origin is used only once per cell cycle

Front 28

PCNA

Back 28

i. Proliferating cell nuclear antigen
ii. Serves as a clamp which is assembled with DNA polymerase delta to ensure processivity
iii. Antibodies to PCNA used clinically to examine degree of cell proliferation in tissue

Front 29

RNA primers are synthesized

Back 29

i. Removed by RNase H

Front 30

Histone and DNA reassociation

Back 30

i. Form old nucleosome structure
ii. Increase in synthesis of new nucleosomes during DNA replication
iii. New nucleosomes are associated with one of the two daughter strands rather than being distributed between bother daughter strands → conservative mechanism of nucleosome synthesis

Front 31

Cyclins

Back 31

i. Initiation of DNA synthesis in S-phase
ii. Control CDKs(cyclin-dependent kinases) at various times in cell cycle
iii. Cyclins A and E specifically control onset of S-phase DNA synthesis

Front 32

CdK2-cyclin E/A

Back 32

i. Phosphorylates pRb
ii. Causes dissocation of hyperphophorylated pRB to activate E2F transcription factor

Front 33

E2F

Back 33

i. Turns on many genes to activate DNA synthesis such as DNA polymerase alpha

Front 34

Inhibitors of DNA replication

Back 34

i. Radiation
ii. Can block activation of CdKs