Unit 1 - Part 2

What is the combined reaction for replication?

(dNMP)n + dNTP + H2O → (dNMP)n+1 + 2Pi

Is replication exergonic or endergonic?

exergonic

semi-conservative replication

for a daughter molecule, one strand is from the parental molecule and the other is newly synthesized

origin of replication

where replication starts on a DNA double helix

What is different about the ori in prokaryotes and eukaryotes and why?

prokaryotes only have one origin of replication while eukaryotes have multiple because prokaryotes have a smaller amount of DNA to be replicated

How are ori of plasmids modified?

often engineered to have really efficient plasmids to interact with proteins that start replication

What are the steps for the initiation of replication in prokaryotes?

1. ori binds to initiator proteins, changing shape of the DNA so that the stretch of DNA by the ori opens up

2. this causes the A-T rich region to unwind

3. helicase separates strands of DNA at the replication fork by breaking hydrogen bonds

4. single binding proteins attach to single strands to block reforming hydrogen bonds

DNA primase

synthesizes short RNA primer in a 5’→ 3’ direction

Helicase

separates strands of DNA at the replication fork by breaking hydrogen bonds and then continuing to separate the strands during replication

DNA Polymerase III

extends the RNA primers by synthesizing DNA in a 5’→3’ direction on BOTH strands in opposite directions since DNA is anti-parallel

leading strand

strand of DNA that grows towards the fork and is continuous

lagging strand

strand of DNA that grows in the opposite direction of the fork and is discontinuous

DNA Polymerase I

extends the 3’ end of Okazaki fragments to connect fragments while removing RNA primer and replacing it with DNA

exonuclease

removes nucleotides from 5’ and 3’ ends

endonuclease

removes nucleotides from within a strand

How does DNA polymerase I have both 5’→3’ and 3’→5’ exonuclease activity?

because it removes nucleotides ahead of it when it deletes the RNA primer and it also proofreads the nucleotides behind it and deletes them when there is a mistake

DNA ligase

catalyzes a covalent bond between 2 fragments on lagging strand after DNA Polymerase I

positive supercoil

twists in DNA upstream from opening of helix caused by helicase separating the helix that prevents replication

upstream

anything in front of replication fork

downstream

the newly synthesized DNA direction

topoisomerase

removes supercoiling upstream to allow replication to proceed

ciproflaxacin

an antibiotic that inhibits bacterial topoisomerase so bacteria can’t replicate

types of topoisomerase

type 1 and type 2

type 1 topoisomerase

removes supercoils by making a break in one strand of double helix and allowing relaxation of the coils and then re-forming the bond

type 2 topoisomerase

remove supercoils by making a double strand break in the helix and adding negative coils to cancel out the positive coils caused by replication

What is an example of type 2 topoisomerase?

DNA Gyrase

processivity factor

converts a distributive enzyme into a processive enzyme

distributive enzyme

works over a short period and is discontinuous

processive enzyme

works continuously over longer periods

Beta clamp

a dimer processivity factor that encircles DNA template and attaches to Pol III and keeps it on task

How does the Beta Clamp differ between the leading and lagging strand?

the leading strand is relatively continuous and in the lagging strand the beta clamp unclamps for the gaps between Okazaki fragments and then reclamps

How does the Beta Clamp affect the behavior of DNA Pol III?

it makes it highly processive

non-Watson-Crick basepairs

mis-pairing of base pairs

Why are non-Watson-Crick basepairs able to be identified and corrected by Pol III?

because it messes with the helix structures

loop in lagging strand

to let lagging strand Pol III travel in same direction as leading strand

DNA Pol Delta and DNA Pol Epsilon

carry out DNA synthesis at replication fork

What is the prokaryotic equivalent for Pol Delta and Pol Epsilon in eukaryotes?

DNA Pol III

DNA Pol Alpha

synthesizes RNA primer then synthesizes 20-30 DNA nucleotides

What is the prokaryotic equivalent to DNA Pol Alpha in eukaryotes?

DNA Primase except DNA Primase does not synthesize DNA nucleotides

DNA Pol Gamma

synthesizes mitochondrial DNA

PCNA

(proliferating cell nuclear antigen) processivity factor for eukaryotic DNA polymerases (Delta and Epsilon)

What is the prokaryotic equivalent of PCNA in eukaryotic DNA replication?

Beta Clamp excpet PCNA is a trimer and Beta Clamp is a dimer

FEN 1

(flap endonuclease) cleaves DNA/RNA primer by displacing it and DNA Pol Delta fills it back in

What are similarities to DNA replication in prokaryotes and eukaryotes?

• DNA ligase

• helicase

• topoisomerase

• single strand binding proteins

Telomerase

corrects the loss of DNA at telomeres to maintain telomere length

Describe the structure of telomerase

there are 2 subunits: TERT and TER

TERT

telomerase reverse transcriptase (protein portion) that synthesizes DNA repeats (TTAGGG) from internal RNA template

TER

telomerase RNA that acts as a template to encode telomeric repeats

loss of DNA at telomeres

results of primer removal during replication

What happens to the overhang of the DNA strand once telomerase lengthens it?

it is filled in with typical primers and polymerases but there will still be a gap which is normal because the goal is not to remove the gap, but to make it longer to maintain telomere length

What causes apoptosis?

damage that cannot be repaired such as

• loss of telomeres

• excessive mistakes during replication

• mutation caused by external forces (UV radiation)

What happens to cells during apoptosis?

the insides of cells break down to prevent inflammation which would alert the host then membrane blebbing occurs

membrane blebbing

little pieces of membrane-bound dead cell (after apoptosis) are broken down by phagocytes

types of mutations for DNA replication

• base substitution

• base insertion

• base deletion

• transitions

• transversion

base substitution

nucleotide change that changes DNA sequence

What is an example of base substitutions?

SNPs

base insertion

one nucleotide is added which shifts everything after it

base deletion

one nucleotide is deleted which shifts everything after it

What is an “indel”?

a base insertion or base deletion that lead to frameshifts

transitions

changes from one purine to another or to one pyrimidine to another (stay in same family)

transversion

changes from a purine to a pyrimidine or vice versa (changes family)

nonsynonymous

change in protein outcome after mutation in DNA

What are the types of nonsynonymous mutations?

missense and nonsense

missense mutation

changes amino acid identity at a position

nonsense mutation

changes amino acid encoding codon to a stop codon

synonymous mutation

mutation does not change amino acids in protein

codon bias and its effect on protein production in an external environment

some organisms prefer certain codons over another even though they encode the same amino acid so synonymous mutations can actually play a role in protein production in an external environment

How is DNA mismatch repaired in prokaryotes?

There are methyl groups on the parent strand of DNA, which is used to identify which base is correct if there is a mismatch. Endonucleases cleave phosphodiester bonds at these sites within a strand. DNA polymerases and ligase replace lost DNA and seal the nicks.

What are polymerase II, IV, and V used for?

in DNA repair

photolyase

enzyme that carries out photoreactivation in response to light activation, breaks covalent bonds in thymine dimer

thymine dimer

a common mutation following UV exposure where two thymines on the same strand are covalently bonded together

photoreactivation

a process in both eukaryotes and prokaryotes that repairs thymine dimer

nucleotide excision repair

a process in eukaryotes to repair a thymine dimer where one strand of helix is removed then replaced

What are the steps to nucleotide excision repair?

1. distorted configuration of DNA is recognized by the enzyme complex

2. DNA is separated and single-strand binding proteins stabilize the single strands

3. endonuclease cuts the strand on both sides of the damage and the damaged part is removed

4. gap is filled in by DNA polymerase and sealed with ligase

What enzymes are used for nucleotide excision repair?

helicase, single strand binding proteins, endonuclease, DNA polymerase, and ligase

cockayne syndrome

lack of DNA repair mechanism

xeroderma pigmentosum

disorder caused by lack of one or more DNA repair proteins that results in skin tumors from UV exposure, multiple malignancies, and extreme photosensitivity