DNA Replication/Repair/Recombination (lecture 5)

Question 1

Error rate of procaryote and eucaryote DNA polymerases:

Answer 1

1/10^9 nucleotides changes per replication

Question 2

how was proofen that replication is semi-conservative?

who did it?

Answer 2

=In every replication new single strands are
synthesized, old single strands are preserved

Meselson and Stahl

synthesis of a new strand with dNTPs containing 14 N (light) to a template strand containing 15 N (heavy)
generated DNA showed medium weight

Question 3

Purines and Pyrimidines

Answer 3

Purines: Adenine and Guanine
Pyrimidines: Cytosine and Thymine and Uracil

Question 4

name the reaction of adding nucleotides to the growing DNA strand

Answer 4

Nucleophilic a`ack of the terminal
3’ –OH group on the α-phosphate
of the nucleoMde;
pyrophospate is released

primer strand=growing strand

template strand

Question 5

reason for asymmetric replication?

Answer 5

DNA Pols can only synthesize in 5# to 3# direction because 3’ OH is required for the nucleophilic attack

-> Leading strand: direcMon of synthesis and replicaMon fork are the same
Lagging strand: synthesis and replicaMon fork move in opposite direcMons

Okazaki fragments at lagging strand

Question 6

why primers are required?

features?

Answer 6

because the DNA Pol needs a first 3’ hydroxyl group
(no de novo synthesis but only elongation!)

• primers are RNA!!
• approx. 10bp
• built by DNAprimase

Question 7

what happens to the primers in replication?

Answer 7

RNA primers are removed by RNAse H

the primase has a low fidelity and the DNA-RNA-hybrids are not useful

nick sealing by DNA ligase

Question 8

3 steps for DNA Pol fidelity?

Answer 8

1) watson-crick base-pairing is favored during 5’-3’ polymerization: 1 in 10^5

conformational change of DNA Pol can happen more easily after correct incorporation

2) 3’-5’ exonucleolytic proofreading: 1 in 10^2

excision of rare, instable tautomeric forms of nucleotides (imino-form of cytosine and enol-form of thymine)

3) strand-directed mismatch repair: 1 in 10^2
combined: 1 error in 10^9 nucleotides

Question 9

catalytic sites for synthesis? why?

Answer 9

Separate catalytic sites for synthesis and editing

why??

Question 10

mismatch repair system

Answer 10

an error in the newly made strand is recognized because of incorrect base pairing by mismtach proofreading proteins:
MutS and MutL

the template strand must be recognized:

eu: nicks (in lagging and leading strand!) marks the new strand!
pro: methylation of A in GATC marks the template strand

newly made strand is removed by mismtach proofreading proteins

-> repair DNA synthesis

Question 11

DNA Polymerases in prokaryotes

Answer 11

DNA pol I/II: DNA repair
DNA pol III: synthesis of leading and lagging strand
holoenzyme consists of seven protein subunits

Question 12

DNA polymerases in eukaryotes

Answer 12

DNA pol α, β, γ, δ, ε

DNA pol α: initiation of replication (priming), error prone! (no exonuclease activity)

(the RNA primers are synthesized by primase!)

DNA pol β: DNA repair

DNA pol δ, ε: replicaMon

DNA pol γ: mitochondrial DNA
replication

Question 13

what’s the matter with DNA polymerases and evolution/ maintainence of healthy organisms?

Answer 13

DNA Pol alpha is responsible for initiation of replication starting at the RNA primers

it is error prone as it lacks a exonuclease activity

that’s why mutations cluster at 5’ ends of okazaki fragments (and the leading strand)
-> lagging strand replication shapes the mutational landscape of the genome

Question 14

Accessory proteins that are required for DNA replication?

why?

Answer 14

DNA is a very stable helical double strand -> not accessible for the DNA pol.

1) ATP dependent unwinding of DNA ds->ss

Helicases

2) Stabilization of ss (prevent formation of hairpins and straighten the ss)

Single strand binding proteins (SSB)

3) Synthesis facilitation/ increase of processivity
(reduce dissociation from DNA)

pro: DNA clamp
eu: PCNA
(proliferating cell nuclear antigen)

a clamp loader fixes clamp and DNA Pol to the DNA under ATP hydrolysis

4) relieving of DNA

Topoisomerases I: ss break -> DNA can rotate around the backbone
ATP INdependent!

Topoisomerase II: manages tangles and supercoils
ATP dependent

Question 15

replication in E. coli?

Answer 15

circular genome
one origin of replication
two replication forks moving in opposite direction

replication speed is constant/not regulated:
4.6x10^6bp -> 500-1000bp/second
~40 minutes per genome

Question 16

origin of replication in e. coli?

Answer 16

250bp

contains 11 GATC motifs

Question 17

how is the replication in e. coli timed?

Answer 17

when replication has just strated, the GATC motifs at the origin of replication are hemimethylated

this allows inhibitor proteins to bind

20 mins later, the origin is fully methylated (when enough ressources are present to complete a full replication cycle) and replication can be re-intitiated

Question 18

eukaryotic cell cycle?

Answer 18

S: DNA replication
G2: cell growth
M: mitosis
G0: testing phase, stop of division
G1: cell growth

Question 19

how can one analyse replication speed and origins of replication in eukaryotes?

Answer 19

audioradiography after periodic exposition to 3H-labelled DNA

on photo-emulsion coated slides

Question 20

speed and orgins in eukaryotes?

Answer 20

Speed of synthesis ~50bp/second

replication units comprise 20-80 clustered origins of replication

intervals of 30.200kb between origins within one cluster

bidirectional replication forks as in bacteria

Question 21

how can one analyse the global DNA replication?

which chromatin is replicated when?

Answer 21

DNA mirco-array

EuchromaMn (less condensed) is replicated early in S-phase
HeterochromaMn (condensed) late in S-phase
inacMvated X-chr. (of mammalian females) is replicated later than the acMve one

Question 22

how can you search for ARS?

Answer 22

ARS= autonomously replicating sequences in yeast genome (contain an origin of replication)

clone randomly selected yeast DNA segments into plasmid vector containing a His gene

grow transformed yeast cultured on His selective medium

those plasmids with ARS will show a higher transformation efficiency/ more yeast cultures on the selective medium because they replicated themselves and created a higher concentration

Question 23

function of ARS?

Answer 23

in yeast they ensure effeicient chromosomal replication

however, deletion of single ARS does not affect replication

Question 24

elemets of ARS?

Answer 24

in yeast

whole ARS: approximately 150bp

• ORC-binding sites (origin recognition complex)
• unwinding region: A/T rich, easy to unwind

-Abf1-binding site:
auxiliary protein, facilitates ORC binding

Question 25

consequence of cell cycle for replication?

Answer 25

accessory proteins need to be activated and inactivated sequentially:

this is mediated by cyclin-dependent-kinase (Cdk)

Cdk activates replication

Question 26

is human replication dependent only on origin of replication?

Answer 26

no, human origins of replication seem to be less well definded

deletion in DNA regions far away from the origin (thousands of bp away) can inactivate an origin

possibly chromatin structure has major influence on the function of origins of replication

Question 27

method to analyze replication in human genome?

Answer 27

whole genome sequencing (WGS)

Sequencing during S-phase
-> Amount of sequence reads correlates with replicaMon state
Early replicaMon->many reads, late replicaMon few reads
Polymorphisms in origins affect efficiency