MCB2021F structure of DNA double helix

reason #1 that DNA structure is stable

hydrogen bonds between complimentary bases and between sugar phosphate backbone and H2O

reason #2 that DNA structure is stable = electrostatic interactions

negatively charged phosphate groups repel one another and interact with Mg2+

reason #3 that DNA structure is stable = van der waals and hydrophobic interactions

stacking of base pairs

how many bonds do A and T share

2 H bonds

how many bonds do G and C share

3 H bonds

which regions of DNA are more stable

G and C

helix is anti-parallel meaning

sugar-phosphates outside and bases stack inside

helix dimensions

10 bp per turn

0.34nm bp spacing

2.37nm diameter

3.4nm pitch

tops of bases line

floor of the major groove

bp edges nearest to the glycosidic bond form

interior surface of minor groove

major groove can accomodate

a protein

regulatory proteins can recognize

pattern of bases and H-bonding possibilities in major groove

what happens when heating DNA >80C

bp interactions disrupted

why does denaturation increase UV absorbance

pi-electrons of unstacked bases

midpoint of absorbance increase

the melting temperature

DNA can also be denatured using

an alkali and/or pH

when temperature is lowered DNA

absorbance drops = re-establishment of stacking

why do DNA's differ in TM values

due to relative G:C content

higher g:c content of DNA results in

higher Tm because G:C pairs have more bonds

what does DNA assume

circular higher order structure

plasmid DNA

bacterial extrachromosomal DNA - closed DNA duplexes

supercoiled state

circular DNA sometimes has more or less than 10 bp per turn

What do topoisomerases/gyrases do?

introduce or remove supercoils

E.coli chromosomal DNA bp

4.64*10^6

length of e.coli chromosome

1.6mm

length of e.coli

0.002mm

how does the chromosome fit in the bacterial cell

supercoiling extensively packages circular DNA to fit

diameter of a typical human cell

20um

genetic material consists of

23 pairs of dsDNA in the form of chromosomes

total length of human DNA

2 meters

How much is DNA condensed?

x100000

how DNA condensation made possible

by wrapping DNA around nucleosomes and then packing them with DNA into helical filaments

chromatin

nucleoprotein complex of DNA

chromatin protein constituents

histones and non-histone chromosomal proteins

what are the 4 pairs in the histone octamer structure

H2A, H2B, H3 and H4

what are the regulators of gene expression

the lesser known histone proteins

what gives rise to chromosomes

higher-order structural organization of chromatin

why does replication of DNA give identical progeny molecules

because base-pairing is the mechanism determining the nucleotide sequence synthesized in each of the new strands during replication

each original strand acts as what for the new strand?

template

what happens with the template strand

it is used to from a new complementary strand by enzyme DNA polymerase

How is DNA replication semi-conservative?

It creates two strands DNA each with 1 new strand and 1 original strand

Where does DNA replication occur?

origins of replication

which way is replication directed

bi-directional = 2 replication forks

What end are nucleotides added to?

3' end

which way does replication occur

5' to 3'

why must double helix be unwound by helicases

to expose single strands

leading strand copies

continuously

lagging strand copies

in segments which must be subsequently join

what happens at the origin of replication

Helicase separates the strands of DNA.

what can unwinding result in

introduction of positive supercoils

gyrase introduces

negative supercoils using atp hydrolysis, this relaxes positive supercoils and seals breakage

single strand DNA binding proteins

bind to ssDNA and stops strands from binding again

DNA primase

primer required to bind at ori

primers

short sequences of RNA

primase

synthesizes primers

DNA polymerase 3

makes new strand by reading template strand and adding 1 nucleotide after the other

leading strand replication

fork moves 5' to 3'

parent antisense strand acting as template for continuous synthesis

lagging strand

replication fork moving right to left

parent sense strand acting as template for discontinuous lagging strand synthesis

DNA polymerase

enzymes that replicate DNA

pol 1 needs

all 4 deoxynucleotides

a template

a primer

pol 1 has 3 active sites

has polymerase activity

has proofreading and editing function

pol 3 is the chief DNA-replicating enzyme of E.coli

sits at each replication fork

why does DNA pol1 recall RNA primer

to initiate strand synthesis

what does DNA pol1 do

replaces RNA primers with DNA during replication using 5' to 3' polymerase activity

what does pol 1 polymerise

about 200 bases before it dissociates from template

repair functions of pol 1

has 3'-5' exonuclease function

has 5'-3' exonuclease activities

why the 3'-5' exonuclease activity

enhances accuracy of DNA replication

3' exonuclease activity

removes nucleotides from 3' end of growing chain

proofreading function of 3' to 5' exonuclease activity

removes incorrectly matched bases. enhances fidelity of replication process

alpha subunit

polymerase

epsilon subunit

3' to 5' exonuclease

theta subunit

stabilization of epsilon subunit

dimeric polymerase

1 unit synthesis leading strand and lagging strand each

beta subunit

forms a sliding clamp around DNA so that pol can move along

clamp loader

responsible for adding and anchoring beta subunit's core structure

replisome consists of

dna unwinding proteins

priming complex

DNA polymerase 3 holoenzyme comprising 2 replicative polymerases

initiation

DNA protein binds to repeats in ori, initiating strand separation

primase binds and constructs RNA primer

elongation

DNA gyrase relieves supercoiling

DnaB unwinds DNA

SSB binds to keep strands separated

DNA pol 3 replicates each strand

termination

ter locus opposite of ori, rich in G and T consensus sequence signals end of replication

tus protein

a contrahelicase that prevents further unwinding

how does tus protein work

blocks helicase and replication fork

lagging strand replication

DNA pol 2 is 1 enzyme with 2 units for leading and lagging

lagging strand is looped around and replication occurs 5 to 3

DNA pol 3 unclamps and reclamps periodically on lagging strand when it encounter primer of okazaki fragments

DNA pol1 eexcises RNA primer and replaces it with DNA

ligase seals remaining nick

DNA polymerase alpha

initiation of nuclear DNA replication, processivity = 200 nucleotides

DNA polymerase epsilon

leading and lagging strand synthesis, checkpoint control

DNA polymerase sigma

principal polymerase in leading and lagging strand synthesis; highly processive

multiple origins of replication

2 replication forks at each origin

mutations that arise due to environmental factors or endogenous errors during synthesis

deletions

insertions

substitutions

replication errors

base mismatches

UV-light

chemical mutagens

integrity of DNA is vital to cell survival and reproduction

repair systems to correct DNA damage

types of substitution mutations

transition and transversion

transition

purine to purine or pyrimidine to pyrimidine

transversion

purine to pyrimidine or pyrimidine to purine

insertions and deletions

insertion or deletion of one or more bases

deamination of cytosine

forms uracil which base pairs with adenine. incorrect base-pairing: C-G becomes U-A

deamination of adenine

forms hypoxanthine which base pairs with cytosine. incorrect base-pairing: A-T becomes C-G

depurination

loss of purines from DNA resulting from hydrolysis of the glycosidic bond between deoxyribose and the base, leaving an apurinic site in DNA

in amino-imino tautomers

an amino group, usually protonated, can tautomerise to an imino group and become deprotonated

when an imino tautomer of adenine base-pairs with cytosine

A-T pair changes to mismatched A-C pair = point mutation, non-coding

what does ultraviolet light promote

formation of covalent bonds between adjacent thymine residues in a DNA strand

what does it mean if a thymine dimer cannot fit into a double helix

replication and gene expression are impaired

chemical mutagens

chemical compounds and alkylating agents