L5: DNA Replication

Question 1

Eukaryotic Cell Cycle

Answer 1

• S phase or DNA Replication

Question 2

Replication

Answer 2

• carefully regulated by the timing of the cell cycle and critical cell cycle “checkpoints”

Question 3

Watson-Crick semiconservative hypothesis

Answer 3

• one of each parental DNA strands will be present in each of the two newly synthesized daughter dsDNA

Question 4

conservative vs. semiconservative DNA replication

Answer 4

• conservative:
  
  • both parental DNA strands are conserved in the daughter DNA
• semi-conservative:
  
  • only one of the two parental strands is conserved in the daughter dsDNA

Question 5

Conservative DNA replication

Answer 5

• both parental DNA strands are conserved in the daughter DNA

Question 6

Semiconservative DNA

Answer 6

• only one of the two parental strands is conserved in the daughter dsDNA

Question 7

DNA replication is ______

Answer 7

semi-conservative

Question 8

5 Features of DNA replication

Answer 8

1. DNA replication starts from a replication origin
2. DNA replication is a DNA template-dependent polymerization process that needs RNA primers
3. DNA synthesis is always 5’-3’, so replication of dsDNA has to process in opposite directions (bidirectional replication)
4. DNA is synthesized continuously (leading strand), as well as discontinuously (lagging strand-synthesized as Okazaki fragments)
5. DNA replication is very accurate and fast

Question 9

Direction of DNA synthesis is ______

Answer 9

• 5’-3’

- the 3’ hydroxyl group of the existing DNA is where the incoming nucleotide will be added

Question 10

DNA synthesis

Answer 10

• substrates: Deoxyribonucleoside triphosphates

- chemistry: 3’ OH attacks phosphate on incoming nucleoside triphosphate

Question 11

Origin of replication

Answer 11

• prokaryote:
  
  • there is one single origin per genome (or plasmid), also called a replicon
• eukaryote:
  
  • have multiple origins in each chromosome (10,000 in human genome)

Question 12

The parental dsDNA needs to be opened to become two ssDNA at the _____ _____

Answer 12

replication origin

Question 13

Replication of a circular bacterial DNA occurs from _____ _____

Answer 13

one origin

Question 14

In _____, there are many origins of replication per chromosome

Answer 14

eukaryotes

Question 15

DNA replication is _____

Answer 15

bidirectional

Question 16

DNA replication is bidirectional

Answer 16

• REPLICATION FORK
  
  • the place where dsDNA “melts” into two ssDNA, there are 2 forks per origin
  • replication process at both ends of the replication fork
• LEADING STRAND
  
  • strand that is synthesized continuously
• LAGGING STRAND
  
  • strand that is synthesized discontinuously, resulting in Okazaki fragments

Question 17

Replication fork

Answer 17

• the place where dsDNA “melts” into two ssDNA, there are 2 forks per origin

Question 18

Leading strand

Answer 18

• strand that is synthesized continuously

Question 19

Lagging strand

Answer 19

• strand that is synthesized discontinuously, resulting in Okazaki fragments

Question 20

Since DNA synthesis is always 5’, one daughter strand is synthesized _____ and the other is synthesized _____

Answer 20

continuously, discontinuously

Question 21

Consequences of replication fork structure

Answer 21

• one DNA strand (leading strand) grows continuously
• one strand (lagging strand) is synthesized in short pieces (Okazaki fragments) that must be joined together
• DNA lagging strands must be initiated repeatedly by RNA priming (primase can initiate an RNA chain)

Question 22

Key enzymes and accessory proteins involved in DNA replication

Answer 22

• helicase
• DNA topoisomerase
• primase
• DNA polymerase
• RNase H
• DNA Ligase
• ssDNA binding protein (SSB)
• sliding clam protein

Question 23

_____ unwinds dsDNA to create ssDNA

Answer 23

Helicase

Question 24

Unwinding of the double helix at replication fork causes _____ of the DNA helix

Answer 24

supercoiling

Question 25

DNA topoisomerase

Answer 25

- removes supercoils by creating a transient break in one or two strands of DNA, which releases the tension and allow DNA to return to normal helix (10bp per turn)

Question 26

_____ is an RNA polymerase

Answer 26

Primase

Question 27

Primase

Answer 27

- an RNA polymerase | - synthesizes short RNA primers (5-10 nucleotides long) to help the initiation of DNA replication

Question 28

SSB

Answer 28

single strand DNA binding protein

Question 29

SSB

Answer 29

- bind and stabilize ssDNA

Question 30

DNA polymerase

Answer 30

- both prokaryotic and eukaryotic cells have multiple DNA polymerases - DNA polymerase III is the major E. coli DNA replication enzyme - has 3 major enzyme activities

Question 31

Major enzyme activity of DNA polymerase

Answer 31

1. 5' -> 3' polymerase activity--for SNA synthesis 2. 5' -> 3' exonuclease activity--for removal of RNA primers 3. 3' -> 5' exonuclease activity--for repairing mistakes

Question 32

RNase H

Answer 32

- specifically degrades RNA in the RNA:DNA hybrid | - removal of the RNA primer by RNase H and the 5'-3' exonuclease activity of DNA polymerase

Question 33

DNA ligase

Answer 33

- creates a phosphodiester bond between an adjacent 5' phosphate and 3' OH - fills the gaps between Okazaki fragments to make a continuous DNA molecule

Question 34

Sliding clamp protein (Beta clamp)

Answer 34

- helps polymerase to bind to the template strand rapidly after each round of lagging strand synthesis

Question 35

Proof reading

Answer 35

- DNA polymerase has proofreading function (3'-5' exonuclease activity), which allows it to detect mistakenly incorporated nucleotides, remove them, and replace with the correct nucleotides - good but not perfect, reason for "spontaneous" mutations in the genome and why organisms have kept evolving

Question 36

Summary: DNA is synthesized in a _____ manner

Answer 36

semi-conservative

Question 37

Summary: DNA is synthesized _____

Answer 37

bidirectionally

Question 38

Summary: At each replication fork, DNA is synthesized continuously on the _____ strand and discontinuously on the _____ strand

Answer 38

leading, lagging

Question 39

Summary: _____ is used as primer

Answer 39

RNA

Question 40

Summary: RNA primer is removed later and replaced by _____

Answer 40

DNA

Question 41

Cancer

Answer 41

- accumulation of multiple mutations in two types of genes and the break-down of regulatory mechanisms of cell growth - two types of genes involved in cancinogenesis are: oncogenes and tumor suppressor genes - mutation in both the proto-oncogene and tumor suppressor gene are usually needed to cause cancer in which also why it is so difficult to treat cancer - tumor suppressor genes are genes for which the loss of function causes cancer. - can encode transcription factors cell-cycle regulators,phosphates, etc... - tumor suppressor gene products are so often inhibitors of cell proliferation

Question 42

Two types of genes involved in cancinogenesis are:

Answer 42

oncogenes and tumor suppressor genes

Question 43

_____ _____ genes are genes for which the loss of function causes cancer.

Answer 43

tumor supressor genes

Question 44

tumor suppressor gene products are so often inhibitors of ____ _____

Answer 44

cell proliferation