Lecture 9 - DNA Replication and Repair

Question 1

What are and explain the 3 proposed models of how DNA replication works

Answer 1

1) Conservative - the two parental DNA strands would re-basepair with each other after being used as templates to synthesize new strands, same with the two new strands being together
2) Semi-conservative - each of the two parental DNA strands would act as a template for new DNA strands to be synthesized, but after replication, each parental DNA strand would basepair with the complementary newly-synthesized strand, and both double-stranded DNAs would include one parental or “old” strand and one daughter or “new” strand
3) Dispersive - after replication both copies of the new DNAs would somehow have alternating segments of parental DNA and newly-synthesized DNA on each of their two strands

Question 2

Describe bacterial DNA replication

Answer 2

Begins at replication origin (replication forks), proceeds bidirectionally (theta replication), once finished, the linked circles are separated by topoisomerase, new cell walls are built

Question 3

Explain the differences between DNA replication in bacteria vs humans

Answer 3

Humans are slower at replicating but have so much more DNA, due to this we have many more points of origin to replicate more DNA

Question 4

Describe eukaryotic replication

Answer 4

It has multiple sites of origin, multiple replication units (replicons), each replicon contains an origin of replication

Question 5

Describe briefly the steps of eukaryotic DNA replication

Answer 5

Replication begins and a point of origin, replication bubbles form, and then the bubbles/replicons fuse together to form four linear strands (2 old/2 new)

Question 6

Describe pre replication in prokaryotes

Answer 6

DnaA protein binds to the 9-mer region, forcing unwinding of the 13-mer region, then DnaC brings DnaB to the 13-mer region to initiate helicase activity

Question 7

Describe the pre-replication complex in eukaryotes

Answer 7

Origin recognition complex (ORC) binds to replication origin (multi-subunit protein complex), then minichromosome maintenance (MCM) proteins bind, these contain helicases that unwind DNA, and helicase loader proteins bind and help MCM bind to the ORC

Question 8

How is licensing regulated?

Answer 8

Cdk’s (protein kinases) are activated during early S phase and active DNA synthesis at licensed origins and prevents origins from being licensed again, they phosphorylate ORC and helicase loader protiens
Geminin blocks the binding of MCM proteins to DNA, after cell division, geminin is degraded and Cdk is inhibited

Question 9

What do DNA polymerases do?

Answer 9

Add new nucleotides complementary to the template strand at the 3’ OH of the growing strand

Question 10

What do primases do?

Answer 10

Add short RNA primers to form short RNA/DNA duplexes

Question 11

What do topoisomerases do?

Answer 11

Reduces supercoiling and prevents twisting of DNA

Question 12

What do helicases do?

Answer 12

Unwind the DNA

Question 13

What do single strand binding proteins (SSB’s) do?

Answer 13

Binds DNA to keep in unwound

Question 14

Which way does DNA polymerase synthesize DNA in?

Answer 14

5’ - 3’
5 in prokaryotes
12 in eukaryotes

Question 15

What happens during DNA synthesis?

Answer 15

Phosphodiester bond if formed between 3’ OH and 5’ phosphate

PPi is released, this provides energy for synthesis

Question 16

What are okazaki fragments and why do they occur?

Answer 16

Short pieces of newly synthesized DNA on the lagging strand, they happen because DNA polymerase only works in the 5’-3’ end, so it needs to move backwards essentially and make it in pieces

Question 17

Name and give the function of the 2 RNA primers in DNA replication

Answer 17

DNA polymerase III adds nucleotides to the 3’ end of an existing nucleotide chain
DNA polymerase I has a 5’-3’ exonuclease activity to remove RNA and fill in gap when DNA reaches an RNA primer

Question 18

Steps in DNA replication with all the components

Answer 18

Initiator protein binds to dsDNA (slight unwinding), DNA helicase (continues unwinding), DNA gyrase (relaxes supercoils), SSB (stabilizes single strands), Primase binds (synthesizes RNA primer), DNA polymerase III (adds nucleotides 5’-3’ direction, RNA primer made for lagging strand, DNA polymerase III extends), Discontinuous synthesis of lagging strand (multiple primers and okazaki fragments), DNA polymerase I (removes RNA primers), then DNA ligase (links okazaki fragments)

Question 19

What is the replisome?

Answer 19

Proteins involved in DNA replication are all closely associated in one large complex

Question 20

What additional proteins do eukaryotes have compared to prokaryotes?

Answer 20

Replication factories synthesize DNA as chromatin fibers are fed through them
Chromatin remodeling proteins loosen nucleosome packing ahead of the replication fork

Question 21

What is the problem at the end of replication?

Answer 21

There is no 3’OH to add nucleotides to at the end of linear DNA. It cannot properly replicate and there is not enough room for another okazaki fragment so the DNA strand gets shortened

Question 22

What is the mechanism for protecting the shortening of DNA?

Answer 22

Telomeres (protective buffer) - is a highly repeated DNA sequence (TTAGGG), its non-coding, this ensures that any loss will not be from an important sequence

Question 23

What is the enzyme called that synthesizes telomeres?

Answer 23

Telomerase - its an RNA/protein hybrid, its not active in all cells, it acts as a template to create more telomeres, also recruits capping proteins to protect the end of the telomere

Question 24

What is the function of telomerase?

Answer 24

RNA matches telomere repeat, its positioned so the enzyme can extend the 3’ end
It adds more copies to the top (longer) strand and makes room for another okazki fragment on the lower (shorter) strand

Question 25

What does exonuclease activity refer to in proofreading?

Answer 25

It cuts the last nucleotide in the sequence if its wrong and makes it right

Question 26

Describe the proofreading mechanism

Answer 26

DNA polymerase error results in mismatched base pair
Daughter strand rotates out of the polymerase site and into the exonuclease site, exonuclease removes mismatched base
Daughter strand resumes DNA synthesis

Question 27

What are tautomeric shifts?

Answer 27

A reversible change in the location of a hydrogen atom in a molecule that alters it from one to another isomer, allows it to base pair with the wrong nucleotide

Question 28

What are the 3 most common DNA mutations?

Answer 28

Depurination - loss of base (A or G)
Deamination - loss of NH2 (amino group), leads to mispairing
Thymine dimers - covalently bonded complex of two adjacent thymines on a single strand of DNA (due to UV), it blocks replication

Question 29

What are the 3 chemical mutagens and explain what they do

Answer 29

Base analogs - resemble the bases, they get incorporated into DNA and prevent replication after that point
Base modifying agents - DNA base structure gets chemically modified
Intercalating agents - insert themselves in between bases, series of blockages, results in insertions/deletions

Question 30

What are the 2 radiation mutation sources and explain what they do

Answer 30

UV - covalently bonds pyrimidines (thymine dimers)

X-rays - generate reactive intermediates (DNA damage, breaks bonds)

Question 31

What are the 2 different types of DNA excision repair and explain what they do

Answer 31

Base ER - detects problem in single position, DNA glycosylases remove base (1) from sugar, Endonucleases break phosphodiester bonds on either side
Nucleotide ER - UVR proteins detect damage and create breaks in backbone, helicase unwinds the piece, cuts out a big portion of nucleotides (30)

Question 32

What is Xeroderma Pigmentosum?

Answer 32

When people do not have the proper enzymes (missing or doesn’t work) to repair UV damage, thymine dimers form

Question 33

What is mismatch repair?

Answer 33

It detects an improper base, but which strand is wrong?

In E. coli, the old strand is methylated and assumes that one is right and fixes the new strand being synthesized

Question 34

Why is uracil not apart of DNA?

Answer 34

DNA wouldn’t know if it consisted of a U because a deaminated C is U, so if a U showed up in DNA, it wouldn’t know if it was supposed to be there or if it was a mutation on the C

Question 35

What is homologous recombination?

Answer 35

If the DNA molecule from one chromosome is broken, the homologue is used as a template to guide accurate repair, leads to a perfect fix of DNA

Question 36

What is nonhomologous end joining?

Answer 36

Uses a set of proteins that bind to ends of broken DNA fragments and join them together, there are no sister chromatids available and results in a major loss of nucleotides on both sides of the break