BB 451 DNA replication

DNA REPLICATION AND REPAIR

What is the difference between a ribonucleotide and deoxyribonucleotide, chemically?

hydroxyl vs hydrogen on position 2 of ribose

What is the result of this?

DNA is more chemically stable than RNA 

Which bases is DNA made of?

purines: Adenosine and Guanine (A and G)

pyrimidines: cytidine and thymidine (C and T)

How do you tell the 5’ end from the 3’ end of a DNA strand?

5’ has phosphate 

3’ has hydroxyl

What types of bonds occur in DNA?

phosphodiester (b/w phosphate and hydroxyl)

In what direction is DNA made?

5’ to 3’

Why are the bases inside and the phosphodiester backbone outside?

bases (nonpolar/hydrophobic) , phosphate and sugars (polar/hydrophilic)

What takes more energy to break, G-C or A-T? Why would a scientist care?

G-C because 3 hydrogen bonds (A-T only has 2). Important to transcription because we need to be able to separate the two strands

What do we mean when we say DNA is antiparallel?

5’-3’ against 3’-5’

What is the most common form of DNA? What other forms are there and what are the circumstances where we might see them?

most common = B form 

A form: more compact, has more bends

z form: doesn't really have major/minor grooves

A and B right handed, Z left. change in direction may help signal where a gene is 

What are the major and minor grooves?

spaces between backbone in double helix where proteins will 

How many base pairs (bps) per turn in B-DNA?

10.5

How does DNA get compacted?

histones help coil up

What do we mean by semiconservative replication of DNA?

when a DNA molecule replicates, each new double helix consists of one strand from the original 

DNA molecule (the "old" strand) and one newly synthesized strand, essentially "conserving" half of the original DNA in each new copy

What enzyme has the job of making new DNA (generic name for it)?

dna polymerase

What are the leading and lagging strands? Why does replication have to work this way?

leading: continuous (5’ to 3’, right direction)

lagging: made in pieces ( has to go opposite way to be 5’ to 3’)

What is a primer in DNA replication and what is it made of? What enzyme makes it?

short rna strand made by primase that gives polymerase something to start with

What are Okazaki fragments?

fragments made from synthesis of the lagging strand

What direction does DNA polymerase go? What type of bond does it catalyze?

5’ to 3’, catalyzes formation of new phosphodiester bonds

reads the DNA strand to know what bases to put in during replication?

What is elongation?

the process of adding nucleotides to an existing DNA strand to make it longer

What is “proofreading”?

3’- 5’ exonuclease. reads newly created base pairs and removes if mismatched

significant improvement in accuracy

What proteins make replication happen efficiently? What are their jobs?

DNA Polymerase:

The primary enzyme responsible for adding new nucleotides to the growing DNA strand, following the base pairing rules with the template strand. 

Helicase:

Unwinds the double-stranded DNA by breaking hydrogen bonds between base pairs, creating the replication fork. REQUIRES ATP

Single-Strand Binding Proteins (SSBs):

Bind to single-stranded DNA regions to prevent them from re-annealing, keeping them accessible for replication. 

DNA Primase:

Synthesizes short RNA primers on the DNA template, which act as starting points for DNA polymerase to begin adding nucleotides. 

Topoisomerase (DNA Gyrase):

Relieves the tension created by DNA unwinding ahead of the replication fork, preventing formation of knots

DNA Ligase:

Joins Okazaki fragments (short DNA segments on the lagging strand) together by forming phosphodiester bonds. 

Sliding Clamp Protein (PCNA in eukaryotes):

Enhances the processivity of DNA polymerase by holding it onto the DNA template, allowing for continuous synthesis of long DNA stretches

What is the replication fork? How many of them will occur in bacterial chromosomes?

where dna replication happens, only one in bacterial chromosome

DNA pol3 in bacteria has what activities?

elongation and exonuclease

How are knots in DNA replication dealt with or prevented?

topoisomerase

How quickly do bacteria replicate their DNA?

every 20 minutes

What types of topoisomerase exist? Which type is gyrase?

A type 1 topoisomerase cuts only one strand of DNA to relieve supercoiling, while a type 2 topoisomerase cuts both strands of DNA. Gyrase is type 2

What is the importance of single-stranded binding proteins?

prevent strands from coming back together and protects it

What is the job of the sliding clamp in prokaryotes (or PCNA in eukaryotes)?

Enhances the processivity of DNA polymerase by holding it onto the DNA template, allowing for continuous synthesis of long DNA stretches

What is the chemical process of the formation of the phosphodiester bond in DNA elongation?

O from hydroxyl group attacks phosphate, loss of pyrophosphate

What does DNA pol1 do?

DNA pol 1 5’-3’ exonuclease activity to remove rna primers and then fills in gap with dna (5’-3’ replication)

What is supercoiled DNA? Why would it be useful? What enzyme could help it supercoil?

topoisomerase can create tension, makes dna smaller

What are writhe and twist? How do we get the linking number?

Linking number=twist+writhes

twist=turn (10.5 bp)

writhes=when double helix crosses itself

How does polymerase chain reaction (PCR) work? What are the steps? What do you get as a result?

replicates desired stretch of DNA from trace amounts (amplification)

1. separate strands of DNA by heating mixture to near boiling (breaks hydrogen bonds)

2. Add in DNA primers (synthetically made) to flank region of interest and lower temperature so they will attach to DNA

3. warm mixture to allow replication to occur (Taq polymerase special, can survive high heat)

repeat process for 30-40 cycles

Contrast prokaryotic and eukaryotic DNA replication.

Why do you think that eukaryotes have multiple replication forks?

their genomes are significantly larger and more complex than prokaryotic genomes

What is the origin (“oriC”)?

specific sequence of DNA that is recognized by replication proteins

How fast do eukaryotic cells replicate their DNA?

Slower, 24 hours

Why do you think that origins are rich in A-T base pairs?

because there's only 2 hydrogen bonds so less energy to separate

What is the problem that linear chromosomes have at their ends?

lose a piece of DNA during replication because don't have a circular chromosome to go around and “catch” it, DNA caps called telomeres help this by repeating sequences (controlled by the enzyme telomerase, very active in embryonic and stem cells ) lose telomeres as we age

What usually happens when an incorrect base is placed in DNA, or an adduct occurs?

remove or repair mutation, if cant signal to go through apoptosis (cell death)

Are you surprised that these occurrences are more common than mutations? Knowing that mutations create the variety that is so important to evolution, what does that tell you about the enormity of time?

mutations are by definition rare events that introduce new genetic variations, while most variations observed within a population are likely due to existing genetic diversity being shuffled around through processes like recombination during sexual reproduction. This highlights the crucial role of time in evolution, as even small probabilities of mutation, when compounded over vast stretches of time, can lead to significant evolutionary change. 

What do thymine dimers result from? What happens, and how is it repaired?

UV radiation. Two thymines will covalently bond with each other, repaired by photolyase

What is nucleotide excision repair?

Cuts regions of DNA do they can be repaired by polymerase

What is base excision repair?

incorrect base removed (U  not supposed to be in DNA)

What does cytosine deaminate to?

Urical, pairs  with A instead of G 🙁

What does uracil-N-glycosylase do?

removes uracil so wrong base pair doesn't happen 

What is DNA mismatch repair?

a cellular process that identifies and corrects errors in DNA base pairing that occur during DNA replication

What causes the bulge?

improper base matches (different number of hydrogen bonds)

When there is a mismatch, how does the cell know which one is the wrong base?

Methylation allows for mutation recognition, old strand is methylated (at A)  so should be correct

When does recombination occur?

during the prophase I stage of meiosis

What is the tetrad made of?

two chromosomes

What is synapsis?

when chromosomes pair up to make a tetrad

What is chiasma?

where the two chromosomes overlap

What happens when the synaptonemal complex forms?

swap genetic material below chiasma

Why is this called crossing over?

because genes are crossing over from one chromosome to another

How do you think this contributes to the variety acted on by evolution?

 Genetic recombination contributes to the variety acted on by evolution by creating new combinations of alleles through the exchange of genetic material during meiosis, essentially "shuffling" genes and generating diverse offspring with unique traits,