BIOL 200 (DNA)

RL7-RL9

What is the purpose of DNA replication?

to duplicate DNA before cell division so each daughter cell inherits the full genome

Parental strand serves as?

a template for the formation of a new daughter strand (complementary to the daughter strand)

In which direction does the DNA replication follows?

from 5’ to 3’

DNA replication proceeds through which type of mechanism?

semi-conservative mechanism (each DNA molecule has one old strand and new strand)

Explain to me the DNA polymerization (process itself), what is required?

catalyzed by DNA polymerase, requires deoxynucloside 5’ - triphosphates, requires primer, proceeds from 5’ to 3’

What are the different steps of DNA synthesis?

1. DNA unwiding

2. Primer formation 

3. Elongation 

4. Primer removal 

5. Ligation 

Key enzymes in DNA unwiding?

enzymes: DNA Helicase (to open and seperate the two strands), topoisomerase (relieve tension, supercoils)

Key enzymes in Primer formation?

enzymes: primase

What is a primer?

a primer is a short RNA molecule = complementary to a single-stranded region of unwounded DNA

Key enzymes in Elongation?

DNA polymerase 

What will the DNA polymerase do?

extend the primer by adding complementary nucleotides

What are the two strands present in DNA synthesis?

1. leading DNA synthesis

2. lagging strand DNA synthesis

What is the leading strand? What is the lagging strand?

follows movement of the replication fork (5’ to 3’)

must be formed in the opposite direction of the movement of replication fork

How many primers are necessary for each strand?

1. leading strand (1 primer = synthesis will continue)

2. lagging strand (primase going to produce primer every few bases = synthesis discontinued) = done in fragments

What do we call those fragments (synthesized from extended primer) in the lagging strand?

Okazaki fragments

These Okazaki fragments are made of?

RNA (primer) and DNA/DNA polymerase extension (RNA needs to be removed and replaced by DNA) = primer removal step

What enzymes are necessary for ligation?

DNA ligase = joining the DNA fragments together

What is the replisome?

complex molecular machinery that carries out DNA replication

Enzyme that start the process of replication?

CMG helicase = contains accessory subunits Cdc45 and GINS complex (activate the helicase), hexamer of MCM

ring shaped molecule (pore)

binds to the leading strand (unwinds the duplex)

What is a problem that lagging strands have that leading strands do not have?

when the lagging strand is single stranded (during DNA replication), its bases are exposed (higher mutation rates, more prone to breaking)

due to discontinued synthesis

What protein would be useful that resembles topoisomerase in a way

RPA (replication protein A) = binds single-stranded DNA, keeps single-stranded DNA template in optimal conformation for DNA polymerase 

What synthesizes the leading strand in DNA?

DNA polymerase epsilon

3 types of polymerases in eukaryotic replisome

1. Polymerase epsilon

2. Primase/polymerase alpha 

3. Polymerase delta (PCNA complex, not a polymerase but work with Pol. delta)

What is PCNA?

homotrimetric protein that encircles DNA and prevents polymerase epsilon or beta complex from disassociating from the template = they keep synthesizing for longer (longer work possible)

What about Primase/polymerase alpha

• has primase activity = primase forms the RNA component of the primer

• DNA polymerase activity = use of actual polymerase alpha (extends the primer with short DNA segments

What about polymerase beta/PCNA complex

it replaces the polymerase alpha/Primase complex and completes the synthesis of Okazaki fragments = synthesis of lagging strand

What is RFC/PCNA complex?

RFC is the PCNA loader, it opens the PCNA ring and loads it on a primer = polymerase binds to PCNA to start synthesis

Additional activities?

ribonuclease H and FEN-1 = removes RNA primers

polymerase delta = replaces the RNA with DNA

DNA ligase = ligates the DNA fragments together

So in order what are the steps?

• Helicase (unwind the two strands)

• RPA (stabilize separated strands)

• primase + poly alpha (synthesize RNA-DNA primers)

• pol epsilon (synthesize DNA continuously)

• pol. delta (synthesize Okazaki fragments)

• PCNA + RFC (clamp and loader to keep polymerases attached)

• DNA ligase (join fragments)

What are origins of replication?

specific DNA sequences where replication begins

What is ORC and 2 functions?

ORC = origin recognition complex = six subunit protein that

1. mark replication origins

2. load helicase aided by other initiation factors

What is an example of initiation factors?

CDK (cyclin dependant kinase)

MCM helicase is loaded in? and activated in?

loaded in G1 and activated in the S phase = ensuring replication occurs one per cell cycle

How does regulation of CDK help the loading of MCM and its activation?

G1: low CDK levels = helicase is loaded (no helicase activation) (also helped by ORC)

S: high CDK levels = no helicase loading, helicase activation

//RL8

DNA is subject to constant change = damage, examples?

hydrolytic depurination (removal of the purine), cytosine deamination, guanidine oxidation, methylation of adenine

These modifications produce many different modified bases, what could that lead to?

blocking of the activity of DNA polymerases during DNA replication

Define for me mutations?

permanent, transmissible changes (to another generation) to the genetic material of a cell

can occur spontaneously by transposable elements, by errors during replication

What are mutagens?

chemical compounds, ultraviolet (UV) radiation or ionizing radiation that increase the frequency of mutations

Example of mutagen?

carcinogen = agent that causes cancer

What are the implications of mutation rates in health

• fathers who had accumulation of mistakes in DNA (add up/mutations) = higher probability having children with schizophrenia, autism

• rate of mutations in most viruses is much higher than in cells = higher probability of selection of more successful variants

Explain the case of T. dohrnii?

immortal jellyfish that can revert to a sexually immature polyp stage in response to stress or ageing (reduce its mutation rate)

has been found to have multiple copies of MSH2 and DNA Pol alpha and delta which was suggested to lower its mutation rate in somatic cells

Name me the different mechanisms to repair DNA

1. repair of mismatches

2. repair of small modifications (base excision repair)

3. repair of bulky lesions (nucleotide excision repairs)

Describe DNA polymerase in terms of making mistakes?

DNA polymerases have high fidelity = meaning they don’t do a lot of mistake (1 error every 10,000), still pretty very frequent

What activity can undergo to fix those errors made by DNA polymerase during replication?

proofreading exonuclease activity and mismatch repair

When are both occurring?

proofreading exonuclease: immediate (during DNA replication)

MMR: post-replicative backup

Which DNA polymerase have this exonuclease or proofreading activity

DNA polymerase epsolin and delta (not alpha)

Template that needs to be fixed with proofreading exonuclease and MMR?

old strand is fine, the new synthesized strand (new one is wrong)

(NER/BER, either one of the strands can be damaged)

Explain to me the mismatch repair mechanism?

• MSH2 + MSH6 = binds to daughter strand (detect lesion)

• triggers the activity of protein MLH1 endonuclease + PMS2 = makes cuts

• DNA helicase = peels off the segment generated by MHL1 + PMS2 (not the same DNA helicase as the one used in DNA replication)

• DNA exonuclease = digest the fragments

• gap repair by polymerase delta and dna ligase

Explain to me the process of Base excision repair (BER)

• DNA glycosylase hydrolyzes the bond between the mispaired base and the sugar phosphate backbone (ribose) = detects the wrong base and remove it /backbone intact

• provides specificity in repair (different types of glycosylase for each different wrong base)

• APE1 cuts inside the DNA backbone where there is no base (ribophosphate backbone)

• Ap lyase (pol.beta) = removes the deoxyribose phosphate

• DNA polymerase beta fills the gap and DNA ligase seals the nick in the sugar-phosphate backbone

Explain to me the process of bulky lesions (nucleotide excision repair) = NER

• complex formed by 23B and XP-C = initial recognition of DNA lesion

• recruit of transcription factor (TFIIH) = unwinding DNA, which then recruits RPA and XP-G (also unwinding activity)

• XP-F is recruited and XP-F and XP-G both have endonuclease activities = generates cuts = fragments removed and degraded

• gap is filled by DNA polymerase delta and continuously joined by DNA ligase

What are the mistakes that NER/BER deal with?

BER/NER deals with DNA that is chemically altered or distorted, (ex: normal bases get incorporated but gets damaged, cytosine deamination)

What is one common thing in the 3 mechanism DNA repair?

required that DNA is in a double-stranded form

When DNA lesions are not repaired, what will the DNA polymerase do?

it will stop it’s activity after encoutering the lesion = preventing completion of DNA replication (doesn’t generate a copy of the genome to the next generation, cell death)

What mechanism do we use in that case (to continue DNA replication even with a lesion/modification, prevent cell death)? what does it involve?

translesion synthesis, use TLS polymerases, bypass the lesion using the damaged DNA as a template

Benefit and consequences?

benefit = the DNA replication can continue 

consequences = translesions polymerases will often insert incorrect base at the position opposite to the lesion, don’t have proofreading exonuclease activity, error prone