DNA Replication

What is the mechanism of DNA Replication’

it is semi conservative

what type of replication yields an original intact DNA molecule and one entirely newly synthesized DNA

conservative replication

what type of replication yields 2 dna molecules, each with one parental and one newly synthesized strand

semi conservative

what replication yields 2 dna molecules that are hybrids ( or mixtures) of parental and newly syntehsIzed DNA

dispersive replication

DNA polymerases with a numeral are found where?

within prokaryotes

DNA polymerases with greek letters are loacted where?

eukaryotes

with DNA polymerase what are the substrates for it

dNTPS

what does DNA polymerase do?

it catalyzes the extension of DNA strand one dNMP at a time

What direction does the dna polymerase syntehsize in

5’ to 3’ direction

what does the dna polymerase require to work?

a template and DNA/RNA primer

During a DNA synthesis reaction, what type of transfer occurs?

phosphoryl group transfer

During a DNA synthesis reaction,what is required at the active site in the DNA polymerase enzyme?

Mg²+

During a DNA synthesis reaction: ________ of the 3’ nucleotide attacks _______ of incoming dNTP

3’OH; α phosphate

During a DNA synthesis reaction,what is released?

it releases PPi (pyrophosphate)

what does an error in replication do?

it can introduce a mutation into the genome

An error in replication can introduce a mutation into the genome.

What will come from the mutation?

it will be permenant and inherited by subsequent daughter cells

What is the average E. coli mutation

What does the DNA Polymerase active site restrict base pairing to ?

watson-crick-franklin bp

DNA Pol active site restricts base pairing to Watson-Crick-Franklin bp

What does this mean?

that before a nucleotide is even added, the enzyme essentially “checks” whether the incoming base can form the correct hydrogen bonding pattern and fit geometrically with the template strand

DNA polymerase has an active site that is highly specific in shape and chemistry, which only properly accommodates correct Watson–Crick–Franklin base pairs (A–T and G–C). This means that before a nucleotide is even added, the enzyme essentially “checks” whether the incoming base can form the correct hydrogen bonding pattern and fit geometrically with the template strand

What is this known as?

presynthetic error control

DNA polymerase has an active site that is highly specific in shape and chemistry, which only properly accommodates correct Watson–Crick–Franklin base pairs (A–T and G–C). This means that before a nucleotide is even added, the enzyme essentially “checks” whether the incoming base can form the correct hydrogen bonding pattern and fit geometrically with the template strand—this is known as presynthetic error control. Because incorrect bases do not align properly in the active site, they are usually rejected before incorporation.

However, this system is not perfect .. what does DNA polymerase do?

it still inserts the wrong nucleotide- but the fidelity of DNA replication remains extremely high because additional proofreading and repair mechanism act after this step to correct most mistakes

When does a mutation occur during replication

rarely

What is the big picture of the accuracy or replication

Even though DNA polymerase isn’t perfect, the combination of multiple correction systems makes DNA replication so accurate that mutations are rare events, not common ones—only appearing after thousands of replication cycles.

what digests polynucleotide chains

nucleases

which nuclease is specific for DNA

DNase

which nuclease is specific for RNA

RNase

what nucelase breaks a phosphodiester at one end of a polynucleotide chain

exonuclease

what direction does exonucelase work

5’→ 3’ OR 3’→5’

which type of nuclease breaks a phosphodiester bond within a polynucleotide chain

endonuclease

what two forms can an endocnuclease be

sequence independent to sequence specific

what type of breaks can endonuclease do

• single strand (nick)

• double strand break

What is a critical mechanism that ensures the extremely high accuracy of DNA replication by correcting mistakes immediately after they occur.

Proofreading

High-fidelity DNA polymerases have two distinct active sites, each with a different function

What are the active sites

• a catalytic site for DNA synthesis

• a 3’→5’ exonuclease site for removing mis-incorporated nucletides

The DNA strand is then shifted to the second active site, which has 3’ → 5’ exonuclease activity, meaning it removes nucleotides from the end of the strand

What does this site do?

excises the incorrectly paired nucleotide, effectively “backing up” the synthesis.

The DNA strand is then shifted to the second active site, which has 3’ → 5’ exonuclease activity, meaning it removes nucleotides from the end of the strand. This site excises the incorrectly paired nucleotide, effectively “backing up” the synthesis.

What happens after removal

the corrected strand is repositioned back into the catalytic site, and DNA synthesis resumes with the correct base. This proofreading step dramatically reduces the error rate of DNA replication

Truie or False: DNA synthesis is bidirectional

true

What sequence does DNA synthesis begin at

origin of replication

what strands seperate in dna synthesis

Parent strands

what does it mean for dna synthesis to be bidirectional

DNA is synthesized in both directions starting at the origin

DNA is synthesized by DNA Pol at sites called

replication forks

what happens at a replication fork

parent DNA is being used as a template for replication by DNA polymerase

What is parent DNA first unwound by

helicase

Parent DNA is first unwound by helicase. What does it use?

ATP hydrolysis

which strand is has DNA synthesis occurs continuously 5’→3’

leading strand

what strand happens in DNA synthesis occuring discontinuosly

Lagging strand

DNa synthesis occuring discontinously via the lagging strand is in what type of fragments as a series of 5’→3’ reactions

okazaki fragments

Under-winding or over-winding DNA causes torsional stress, what does this result in

the formation of supercoils

What are the enzymes that add/remove supercoils in DNA by cutting phosphdiester bonds in one or both strands, unwrap the helic, then reseal the strands

topoisomerase

Topoisomerases are the enzymes that add/remove supercoils in DNA by cutting what type of bonds in one or both strands , unwrapping the helix, and then resealing strands

phosphodiester bonds

Supercoiling can be helpful in what type of packaging

genome

Supercoiling can be helpful in genome packaging

• what is used in bacteria that introduces negative supercoils to compavct the genome and also removes positive supercoils in front of replication forks

Topoisomerase II (DNA gyrase)

what are antibiotics that target bacterial DNA gyrase

fluoroquinolones

True or false: Fluoroquinolones blocks DNA gyrase (toposiomerase II) ability to reseal DNA

True

What are fluoroquinolones used to treat

to treat urinary tract infections, respiratory infections, and gastrointestinal infections

What type of trait does fluoroquinolones use that is selctive for bacterial enzymes

selective toxicity

Where does bacterial DNA replication initiation occur at?

oriC

When does bacterial replication occur?

only once per round of cellular division

To make sure that replication in prokaryotes occurs only once per roound of cellular deivision- What is the most regulated step in bacterial dna replication

initiation

What is a unique 245 bp sequence in prokaryotes?

the origin of replication “oriC”

What are R1-5 and I1-3 binding sites for?

Used for DnaA proteins to intiate replication

What is an AT-rich segement where strand seperation occurs for bacterial dna replication during initiation

DNA unwinding element (DUE)

What are binding sites for proteins called replication initiation factors

IHF and FIS

What are the steps of E. coli DNA Replication Initiation

1. DnaA binds ATP to become active

2. DNaC binds ATP and loads a DNaB helicase at both ends of the replication bubble

3. DNA polymerase and additional proteins are added to both DnaB helicases

4. Hydrolysis of the ATP bound to DnaA

In the first step of E.Coli DNA replication initiation what happens

DnaA binds ATP to become active

What happens once DnaA binds ATP to become active

DnaA protein binds to oriC→ psotive supercoil→ DNA denaturation at the DUE

What happens once DnaA binds ATP to become active allowing for the DnaA proteins bind to oriC for a postive supercoil and dentauration at the DUE

DnaC binds ATP and loads a DnaB helicase at both ends of the replication bubble

WHat happens once DnaC binds ATP and loads a DNaB helicase at both ends of the replication bubble

helicase leads the replication fork seperating DNA strands 5’→3’ using ATP and DnaC dissociates

_________ and additional proteins are added to both DnaB helicases since a pre-existing single stranded DNA and primer is now available ( which is required for this enzyme)

DNA polymerases

What happens after DNA polymerase and additional proteins are added to both DnaB helicases

Hydrolysis of the ATP bound to DnaA (topoisomerase II)

What happens after hydrolysis of the ATP bound to DnaA

DnaA dissociates and is very slow to release ADP allowing for strict regulation

What regulates initiation

DNA Methylation

What is oriC DNA methylated by ?

Dam methylase

What does Dam mean ?

Dna Adenine Methylation

What does Dam methylate

it methylates N^⁶ position of A within (5’) GATC sequence

What location has a lot of methylated N^6 position of A within (5’) GATC sequence

the origin

What happens after DNA replication

DNA is hemimethylated

What does hemimethylated oriC associated and sequestered with ?

plasma membrane

When can replication begin again

only after the DNA is fully methylated

What catalyzes DNA synthesis

Core polymerase (Pol III)

What serves as:

• a scaffold for DNA polymerase III complex

• to to assemble the β clamp onto DNA using ATP.

• coordinates the replication fork by interacting with DnaB helicase through τ subunits.

the clamp loader

What is the β clamp (sliding clamp) on the Polymerase III holoenzyme ?

it tethers the core polymerase to DNA

What does the β clamp (sliding clamp) decrease ?

it decreases the polymerase dissociation from DNA to increase processivity

What generates primers for Pol III

Primase

What does DNA polymerase III require

primed DNA template (3’-OH)

What is an RNA polymerase

a primase

True or False: Primase is DNA template-dependent

true

True or false: primase is is primer independent?

true

What does primase do?

it synthesizes a <9nt RNA primer at the beginning of the leading strand and each okazaki fragment

What is the basic lagging strand synthesis

DnaB helicase travels along the lagging template strand in the 5’→3’ direction and unwinds the DNA

What travels along the lagging template strand in the 5’→3’ diorection and unwinds the DNA

DnaB helicase

What does DnaB helicase travel along in the 5’→3’ direction to unwind the DNA

the lagging template strand

As DnaB helicase travels along the lagging template strand in the 5′→3′ direction and unwinds the DNA

what binds the single stranded DNA

single strand DNA binding protein (SSB)

As DnaB helicase travels along the lagging template strand in the 5′→3′ direction and unwinds the DNA

what occasionally associates with DnaB helicase and synthesizes a short RNA primer

DnaG primase

As DnaB helicase travels along the lagging template strand in the 5′→3′ direction and unwinds the DNA

where does primase function?

at the replication fork just as core polymerase nears completion of an Okazaki fragment

What happens after:

As DnaB helicase travels along the lagging template strand in the 5′→3′ direction and unwinds the DNA

A new β clamp is loaded onto the lagging strand at each new RNA primer by the clamp loader

Durin glagging strand synthesis what happens after:

A new β clamp is loaded onto the lagging strand at each new RNA primer by the clamp loader

A new β sliding clamp is loaded onto the lagging strand at each new RNA primer by the clamp loader

In lagging strand synthesis part 2 , what happens after:

A new β sliding clamp is loaded onto the lagging strand at each new RNA primer by the clamp loader

• clamp loader binds ATP then sliding clamp

• The clamp loiader opens the sliding clmap at one subunit interface

• ATP hydrolysis closes the clamp around the DNA, and the clamp loader dissociates

In lagging strand synthesis part 2

• after a new β sliding clamp is loaded onto the lagging strand at each new RNA primer by the clamp loader what closes the clamp around the DNA and the clamp loader dissociates

ATP hydrolysis