DNA Replication, Repair and Recombination 1 (Lec 3)

Question 1

What is the error rate in DNA replication?

Answer 1

1 mistake in every 10^9 base pairs.

Question 2

True or False?

Germ cells have to have low mutation rates to maintain the species.

Answer 2

true

Question 3

Why do somatic cells need low mutation rates?

Answer 3

to avoid uncontrolled proliferation/cancer

Question 4

What reaction does DNA polymerase catalyze?

Answer 4

DNA synthesis

(DNA)n residues + dNTP -> (DNA) n + 1 residues + P2O7 ^4-

Question 5

How is the DNA template directed?

Answer 5

new chain is assembled in a preexisting DNA template that is complementary to the incoming bases

Question 6

True or False?

DNA replication requires dATP, dGTP, dCTP and dTTP

Answer 6

true

Question 7

DNA polymerase requires a primer with a free ___ to begin

Answer 7

3’ -OH

Question 8

What is the direction of growth of DNA?

Answer 8

5’ to 3’

Question 9

Explain how the replication fork is asymmetric

Answer 9

Both strands are simultaneously replicated, DNA polymerase can only synthesize DNA in the 5’ to 3’ direction, leading strand is synthesized continuously, lagging strand is synthesized in segments

Question 10

Where does the first step of proofreading occur?

Answer 10

just before a new nucleotide is added: enzyme must tighten its “fingers” around the active site, which is easier if the correct base is in place

Question 11

When does exonucelolytic proofreading occur?

Answer 11

takes place immediately after incorrect base is added

Question 12

What does 3’ to 5’ exonuclease do during proofreading?

Answer 12

clips off unpaired residues at 3’ primer terminus

Question 13

How does 5’ to 3’ replication allow efficient error correction?

Answer 13

high energy bond is cleaved providing the energy for polymerization

Question 14

How is the lagging strand synthesized?

Answer 14

backstitching process: DNA primase synthesizes a new RNA primer, DNA polymerase adds to new RNA primer to start new okazaki fragment, DNA plymerase finishes fragment, old RNA primer erased and replaced by DNA, nick sealed by DNA ligase

Question 15

What is the protein at the DNA replication fork that unwinds DNA?

Answer 15

helicase

note: hydrolizes ATP which causes conformational change that propels it like a rotary engine along single stranded DNA

Question 16

What do single-stranded DNA binding proteins do?

Answer 16

bind tightly and cooperatively to exposed single-stranded DNA; help stabilize unwound DNA, prevent formation of hairpins, and keep DNA bases exposed

Question 17

What is the function of the sliding clamp at the replication fork?

Answer 17

keeps DNA polymerase on DNA when moving; releases when double stranded DNA is encountered

Question 18

What is the difference in the clamp on the leading strand compared to the lagging strand?

Answer 18

leading strand: clamp remains associated to DNA polymerase for long stretches

lagging strand: clamp loader stays close so it can assemble a new clamp at start of each new okazaki fragment

Question 19

In mismatch repair, how are errors detected?

Answer 19

errors are detected based on distortion caused by mispairing

Question 20

What is the process of mismatch repair?

Answer 20

Binding of mismatch proofreading proteins (MutL and MutS) scan for the nick and trigger degradation of nicked strand, the repair is finalized with new DNA synthesis

Question 21

What is DNA Topoisomerase?

Answer 21

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

Question 22

What does Type I Topoisomerase do?

Answer 22

catalyzes the relaxation of supercoiled DNA

Question 23

How does Type I Topoisomerase work?

Answer 23

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

Question 24

What does Type II Topoisomerase do?

Answer 24

makes a transient double-stranded break in the DNA

Question 25

How does Type II Topoisomerase work?

Answer 25

it uses ATP to 1) break double stranded helix reversibly to create "gate" 2) causes second strand to pass through 3) reseals break and dissociates note: prevents severe tangling problems that would arise during DNA replication

Question 26

What is the DNA replication origin?

Answer 26

A-T rich regions where sequence attracts initiator proteins to pry open DNA

Question 27

How is DNA replication in bacteria initiated?

Answer 27

initiator proteins bind to specific sites in origin, forming complex which attracts DNA helicase + helicase loader

Question 28

In regards to DNA replication in bacteria, once helicase loader is finished loading, what happens?

Answer 28

helicase unwinds DNA so primase can make RNA primer on leading strand; remaining proteins assemble to create 2 replication forks with complexes moving in opposite direction with respect to the origin

Question 29

In terms of the cell cycle, where does Eukaryotic DNA replication occur?

Answer 29

during S phase

Question 30

Different regions of each chromosome are replicated in a reproducible order during S phase, depending on ____ ____

Answer 30

chromatin structure ex: heterochramtin is late-replicating ( X chromosomes of females)

Question 31

Why is chromatin structure important for mammalian DNA replication origins?

Answer 31

they can still function if moved to a different locus if placed where chromatin is uncondensed example: distant DNA in beta globin is required for expression of the genes in the cluster

Question 32

Histone proteins are synthesized mainly in what phase of the cell cycle?

Answer 32

S phase

Question 33

As replication fork passes through chromatin, histone octamer breaks into what?

Answer 33

an H3-H4 tetramer - distributed randomly to daughter duplexes 2 H2A-H2B dimers which are released form the DNA

Question 34

Reassembly of the histones requires what type of protein?

Answer 34

chaperones; directed to DNA with sliding clamp called PCNA

Question 35

What does Telomerase do?

Answer 35

replicates chromosome ends; replenishes special sequence at end of chromosome by elongating the parental strand in 5' to 3' direction using an RNA template on the enzyme note: special sequence is GGGTTA

Question 36

After extension of parental strand by telomerase, what happens?

Answer 36

replication of lagging strand can be completed by DNA polymerase, using extension as template

Question 37

What does the telomere replication mechanism ensure?

Answer 37

3' end is longer, leaving a protruding single stand end that loops back and tucks into the repeat (T-loop)

Question 38

How is telomere length regulated?

Answer 38

each chromosome end in a given cell contains variable number of telomere repeats depending on age; after many generations, daughter cells will have defective chromosomes and stop dividing (replicative senescence)

Question 39

Human fibroblasts normally divide how many times before undergoing replicative senescence?

Answer 39

60