Unit 4 - DNA Replication & Repair

A DNA

RNA/DNA hybrid with narrower major grooves, wider minor grooves formed in low relative humdity

A DNA Diameter

26A

A DNA Helical Twist per bp

31 degrees

B DNA Diameter

20A

B DNA Helical twist per bp

36 degrees

Z DNA

Left handed DNA structure formed in high salt and purified DNA

Linkin Number

Number of times DNA winds around the other, can’t be changed as long as both strands remain covalently intact

Twist

Number of complete revolutions

Positive Twist

Right handed duplex

Negative Twist

Left handed duplex

Writhing Number

Number of turns the duplex axis makes around the superhelix axis

Linking Number Formula

L = T + W

Negative Supercoiling

dsDNA underwound causing twisting in opposite direction of natural double helix

Positive Supercoilding

DNA overwound, increasing total linking number

Separation of DNA causes

Twisting, therefore DNA is negatively coiled

Negative coiling helps with

Replication machinery to get access to the strands in replication

Type I Topoisomerase

Cuts a strand of DNA to unwind the DNA once

Type IA

Relaxes negatively supercoiled DNA by cleaving phsophodiester bond, passing the other strand through the break and ligating with energy from breaking the bond

Type IB

Relaxes both supercoils by cleaving a bond and allows the DNA to rotate more than 1 turn before resealing

Type I Topoisomerase Mechanism

1. unprontonated phenol on Tyr to attack the phosphodiester bond

2. Pentacovalent trigonal bypyramidal transition state occurs

3. General acid helps protonate

What intermediate does IA form

5’ phosphotryrosine intermediate

What intermediate does IB form

3’ phosphoryltyrosine intermediate

Type II Topoisomerase

Dimer protein with two active sites that cuts both strands to change writhes by passing a segment through another using ATP via a 5’ phosphoryltyrosine intermediate

DNA Gyrase

Type II topoisomerase in bacteria that introduces additional negative supercoils with ATP

Ciprofloxacin

Acts on DNA gyrase to enhance DNA cleavafe rate or decrease rate of sealing broken DNA

All plasmids isolated from E Coli are

Negatively supercoiled

All relaxed plasmids have

0 writhes

Histone Structure

2 of each H2A, H2B, H3 and H4

Outer Lysine Histone Residues

Allows interaction with negative DNA charge

Heterochromatin

Transcriptionally silent

Euchromatin

Transcriptionally active

Nucleosome

DNA wrapped around histones

Nucleosome aggregate to make

30nm fibre

Nucleosome Super Loops

1um with 0,4um in between the smaller coils

H3 Lys 9 and H4 Lys 16 can be

Acetylated or methylated

Ser Residue Phosphorylation

Creates a bulky negative charge

Methyl Transcriptase

Adds CH₃ onto cytosine on its C5 which helps discern the newly replicated and old strand

How many CpG islands are methylated in mammal

~80%

Enzymatic Methylation of Pyrimidines

C5 doesn’t change base pairing

Cytosine can spontaneously deaminate to

Uracil with a half life of 19 days

5-methylcytosine spontaneously deaminates to

Thymine

Conservative Replcation

Duplicate both strands

Factory Model of Replication

Replication fork is stationary and DNA is reeled through instead

Helicase

ATP dependent donut shaped protein that unwinds DNA helix, and creates positive coils

SBS

Coats DNA strands to protect from nucleases, and DNA forming secondary structure

DNA Polymerase III

Adds bases using a free 3 OH to grab onto

DNA Polymerase I

Replaces primers

Sliding Clamp

Makes DNA polymerase stay on longer before falling off (processivity)

RNase H

Removes RNA primers and some adjoining DNA in okazaki fragments

DNA Addition

5’ → 3’ direction with C3 OH attacking PO₄ on C5 to form a pentacovalent transition state

Pyrophosphatase

Breaks pyrophosphate into 2 PO₄ to push DNA addition to the right and irreversible

What happens to the lagging strand as DNA polymerase adds bases?

Loops to lets DNA polymerase move in the same direction as replication fork

Tom Steitz

Discovered 3D structure of DNA polymerase klnenow fragment (oven mit)

DNA Polymerase Active Site

Has 2 Mg ions coordinated with Asp Acid to neutralize the negatiev charge and enhance 3 OH to become a nucleophile

How far apart are the DNA polymerase Mg Ions

3.6A

Processivity

Average number of bases added before DNA falls off

In Vitro Processivity

~10 to 15 catalytic cycles

In Vivo Processivity

~5000 from sliding clamp

E. Coli Sliding Clamp

B subunit of DNA polymerase III (dimer)

What is the human sliding clamp?

PCNA (trimer)

DNA Mismtach

Misincorporated nucleotide creates a bump and DNA polymerase uses it's 2nd active site 3 → 5’ exonuclease to hydrolyze nucleotide at the 5’ end

Eukaryotic Ligase uses

ATP

Prokaryotic Ligase uses

NAD

Telmorase

Ribonucleoprotein that repeatedly adds a 6 sequence nucleotide with its template