DNA replication

Question 1

Basic properties of DNA replication

Answer 1

1. DNA replication is semi conservative- one strand is the parent strand and one strand is the daughter strand
2. DNA is synthesized in the 5’-> 3’ direction
3. A 3’ OH primer and a template strand is needed
4. DNA replication is semi discontinuous

Question 2

DNA is synthesized in the 5’-> 3’ direction

Answer 2

To synthesize DNA you need to have a template strand and you need a free 3’OH primer on the growing chain to attach a new nucleotide

DNA polymerase catalyzes this even, the 3’OH group attacks the alpha phosphate of the incoming dNTP so for every addition of a nucleotide, you get rid of 2 P

Question 3

DNA replication is semidiscontinous

Answer 3

When you are copying the 3’-> 5’ strand (leading strand) there’s no issue because youre synthesizing DNA in the 5’->3’ direction and there’s always a free 3’ OH group on the growing strand

But when you are copying the 5’->3’ strand (lagging strand) you run into an issue you need an RNA primer (to provide a free 3’OH) via primase that lays down okasaki fragments before DNA polymerase can go on and add nucleotides

Question 4

DNA replication occurs in 4 basic steps

Answer 4

1. separation of the 2 complementary strands at the origin of replication
2. formation of the replication fork (primers and okazaki fragments)
3. chain elongation
4. removal of primers

Question 5

properties of e.coli DNA polymerase 1

Answer 5

one protein that has 3 catalytic activities:

5’->3’ polymerase
3’->5’ exonuclease (proofreading activity)
5’->3’ exonuclease (removal of primers)

Question 6

Proofreading (error correction by 3’->5’ exonuclease activity) of DNA pol 1
steps

Answer 6

1. DNA Pol 1 accidentally puts in a wrong nucleotide
2. DNA pol1 repositions itself so that the wrong nucleotide is in the 3’->5’ exonuclease activity site, wrong nucleotide gets hydrolyzed and removed
3. reposition back to 5’->3’ polymerase activity site
4. DNA polymerase puts in the correct nucleotide

Question 7

Compare and contrast DNA polymerase 1 and DNA polymerase 3

Answer 7

DNA Polymerase 1: 
made from pol A gene
one subunit
relatively small
has 3'->5' exonuclease activity
has 5'->3' exonuclease activity
relatively slow (polymerization rate)
relatively low stamina (processivity) 

DNA Polymerase 3: 
made from pol C and E genes
9 subunits
relatively large
has 3'->5' exonuclease activity
DOES NOT have 5'->3' exonuclease activity
relatively fast (polymerization rate)
relatively high stamina (processivity)

Question 8

Major difference between prokaryotic and eukaryotic DNA replication

Answer 8

eukoryotes have many origins of replication

prokaryotes only have 1

Question 9

Proteins involved in DNA Replication

Answer 9

Helicase: unwinds DNA
Primase: lays down RNA primers (okazaki fragments)
ss DNA binding proteins: keep DNA separated
DNA gyrase/ topoisomerase 2: unwinds the positive supercoils that are caused by the origin of replications
ligase: seals nicks after DNA pol 1 removes and adds
DNA pol 1: 5’-> 3’ exonuclease activity , eats up primer and polymerase

DNA pol 3: lays down newly synthesized DNA with the available 3’ OH primer

Question 10

Maintenance of Chromosome Ends

Answer 10

ends of chomosomes consist of hexanucleotide sequence (GGGGTT)

during replication, each time the chromosome shorten, telomerase helps keep the length by recognizing this seqence

Telomerase:
Has lots of (CCCCAA) its a template for the syntesis,
it extends the 3’ end of the the chromosome and hangs off, it uses its own RNA

RNA dependent DNA polymerase that carries its own RNA template, somatic cells dont really have telomerasse