exam 3 DNA replication and repair

Question 1

what are the six problems that need to be solved during DNA replication

Answer 1

1. strand polarity - have to synthesize both strands at same times, but polymerase only goes one way
2. unzip DNA - separate two strands
3. processivity (the idea that you continue to add nucleotides to a polymer without stopping - long molecules - have to make sure polymerase doesn’t fall off DNA
4. untangle - two strands are wound around each other - will tangle molecules if pulled apart
5. one copy - only make one DNA copy as replicating
6. accurate copy - DNA molecule contains genetic info that is accurate

Question 2

why are there problems during DNA replication

Answer 2

the two strands of DNA are replicated coordinately using different mechanisms but by the same polymerase in the 5’ to 3’ direction

Question 3

why do eukaryotes have additional complications that prokaryotes don’t

Answer 3

they have a linear genome and the presence of histones

Question 4

how is DNA replication semi-conservative

Answer 4

for each daughter strand, one is a parental strand, and one is a newly synthesized strand

Question 5

which way does replication and chain growth occur in

Answer 5

5’ to 3’ direction - new nucleotides are added at the 3’ end

Question 6

what is DNA replication catalyzed by

Answer 6

DNA polymerase - an enzyme with fingers, palm, and thumb domains

Question 7

what is the solution to the first problem - strand polarity

Answer 7

okazaki fragments are synthesized on the lagging strand and DNA ligase seals the gap between successive fragments

Question 8

what is the solution to the second problem - unzip DNA

Answer 8

DNA helicase unzips DNA and uses ATP hydrolysis to drive a rotary engine. unzipped DNA is stabilized by single-stranded binding protein

Question 9

what is the solution to the third problem - processivity

Answer 9

a sliding clamp holds the DNA polymerase in place, then is loaded on DNA by a clamp loader that uses ATP hydrolysis to lock the clamp around DNA

Question 10

where do the first three problems occur

Answer 10

at the polymerase complex because they’re all assembled on DNA

Question 11

what is the fourth problem - the unwinding problem

Answer 11

rapid rotation of DNA is needed ahead of the replication fork - strands are pulled apart, inducing torsional dress on DNA

Question 12

what relieves torsional stress

Answer 12

topoisomerases

Question 13

what are the two classes of topoisomerases

Answer 13

nick and swivel mechanism (I) and gating mechanism (II)

Question 14

what occurs with topoisomerase I

Answer 14

it covalently attaches to a DNA phosphate, breaking a phosphodiester linkage in one DNA strand. now the two ends of the double helix can rotate relative to each other, relieving torsional stress

Question 15

what is/isn’t required for topI***know!!

Answer 15

• no ATP is required to relieve strain

- a single-stranded nick/break in DNA is required, as is a covalent attachment to the enzyme

Question 16

what occurs with topoisomerase II

Answer 16

binds to DNA in two places - each other then binds to ATP and induces a double-stranded break in DNA

Question 17

once topII induces a double-stranded break in DNA, what happens

Answer 17

second DNA helix can be passed through - method cuts DNA, undoes knot, and reseals DNA after - enzyme is gate to let second piece of DNA pass through

Question 18

what is required to use topII

Answer 18

ATP to untangle DNA and ATP hydrolysis to reseal DNA after

Question 19

how does topII cut linked rings

Answer 19

topII separates linked rings by cutting DNA and allowing other ring to pass through it - topII is required in bacterial cells to complete replication

Question 20

how are topI and topII different relieving stress wise

Answer 20

• topI requires that DNA unwinds along its length, which takes time
• topII can relieve stress more rapidly bc it’s making the cut but comes at cost of ATP

Question 21

why do you need topII any time you have a circular piece of DNA

Answer 21

topI cannot allow two linked rings to pass each other, but topII can - any time you have a circular piece of DNA that’s replicated, need topII

Question 22

what do enzymes topI and topII have in common

Answer 22

both have nuclease activity

Question 23

what is the solution for problem 5 (prokaryotic)

Answer 23

prokaryotic origins have a refractory period due to methylation - prevents restarting replication until cell division has happened

Question 24

what is methylation in prokaryotic cells mediated by

Answer 24

Dam methylase - once original replication is complete, metylase is re-expressed and newly synthesized strand can be methylated and replication can occur again

Question 25

what are the two accessory proteins the origin replication complex recruits in eukaryotic cells

Answer 25

Cdc6 and cdt1 - allow two helicases to bind and go in opposite directions. cdc6 gets degraded eventually to release helicases to unwind DNA

Question 26

what is the solution for problem 5 (eukaryotic)

Answer 26

kinase allows DNA replication to start and prevents it from starting a second time - in order to restart replication, must get rid of kinase, reset system, and get kinase back again

Question 27

what does telomerase prevent

Answer 27

linear DNA ends from being lost during replication