Lecture 3: DNA Replication, Repair and Recombination 1

Question 1

Why do multicellular organisms need high fidelity?

Answer 1

• germ cells need it to have low mutation rates to maintain the species
• somatic cells ned low mutation rates to avoid uncontrolled proliferation/cancer

Question 2

What are the requirements of DNA replication?

Answer 2

• separation of the two parental strands
• dATP, dGTP, dCTP, and dTTP
• DNA polymerase requires a primer with a free 3’ OH to begin

Question 3

When does exonucleolytic proofreading occur?

Answer 3

immediately after incorrect base is added

Question 4

What happens during exonucleolytic proofreading?

Answer 4

• DNA polymerase requires a perfectly paired 3’ terminus

- 3’ to 5’ exonuclease clips off unpaired residues at 3’ primer terminus

Question 5

What is DNA helicase?

Answer 5

• unwinds DNA

- 6 identical subunits that bind and hydrolyze ATP

Question 6

What are single stranded binding proteins?

Answer 6

-bind highly and cooperatively to exposed SS DNA

Question 7

What does single stranded binding proteins do?

Answer 7

• help stabilize unwound DNA
• prevent formation of hairpins
• DNA bases remain exposed

Question 8

What does the sliding clamp do?

Answer 8

Keeps DNA polymerase on DNA when moving, releases when double stranded DNA is encountered

Question 9

Assembly requires a _______ which _______

Answer 9

clamp loader; hydrolyzes ATP as it load the clamp onto a primer template junction

Question 10

What does the clamp do with the leading strand of DNA?

Answer 10

clamp remains associated to DNA polymerase for long stretches

Question 11

What does the clamp do with the lagging strand?

Answer 11

clamp loader stays close so it can assemble a new clamp at start of each Okazaki fragment

Question 12

What does mismatch repair do?

Answer 12

removes (almost all) errors missed by proofreading by detecting distortion caused by mispairing

Question 13

What is DNA topoisomerase?

Answer 13

reversible enzyme that breaks a phosphodiester bond to change superhelicity, relieving supercoiling

Question 14

How does type I topoisomerase work?

Answer 14

creates single strand break in DNA which allows the DNA on either side of the nick to rotate freely relative to each other; uses phosphodiester bond as a swivel point

Question 15

Which type of topoisomerase is thermodynamically favorable?

Answer 15

Type I

Question 16

How does type II topoisomerase work?

Answer 16

Makes a transient double strand break. Uses ATP to:

• break one double stranded reversibly to create gate
• causes second stand to pass through
• reseals break and dissociates

Question 17

What is a replication origin rich in?

Answer 17

A-T sequences

Question 18

What does it mean by the refractory period referring to replication origins?

Answer 18

delay until new strand in methylated

Question 19

Minimum requirements for sequence to be ORI

Answer 19

• binding site for origin recognition complex (ORC)
• A-T rich stretch for easy unwinding
• binding site fro proteins that help attract ORC

Question 20

What is required for chromatin reassembly after replication?

Answer 20

histone chaperones

Question 21

What sequence is at the end of chromosomes and repeated?

Answer 21

GGGTTA

Question 22

What do telomerase do?

Answer 22

replenish end sequences by elongating parenting stand in 5’ to 3’ direction using an RNA template on the enzyme

Question 23

____ cells retain full telomerase activity.

Answer 23

stem

Question 24

What is replicative senescence?

Answer 24

when daughter cells have defective chromosomes and stop dividing; in this way the cell’s lifetime is regulated to guard against cancer