Week 3 - Eukaryotic DNA Replication

differences between eukaryotes and prokaryotes

The DNA of eukaryotes is enclosed in a nucleus

Prokaryotes lack a nucleus

eukaryotes

consists of a true nucleus with a nuclear membrane and nucleoli; genetic material DNA is present inside the nucleus

Genome

All the DNA of an organism is referred to as an organism

Prokaryotic DNA

Usually consists of a single circular chromosome

Found in the nucleoid region (no membrane-bound nucleus)

Typically has one origin of replication• Circular structure means no chromosome ends

Eukaryotic DNA

Organized into multiple linear chromosomes

(Example human -46 chromosomes, dog -78 chromosomes)

Located inside a membrane-bound nucleus

Each chromosome contains multiple origins of replication

Linear structure creates chromosome ends (telomeres)

DNA in both the nucleus and mitochondria, but the nuclear DNA represents the genome

Replication of the genome occurs in the nucleus

nucleolus

Found inside the nucleus and produces ribosomes

chromatin

Substance found in eukaryotic chromosomes that consists of DNA tightly coiled around histones

nuclear pore

a protein-lined channel in the nuclear envelope that regulates the transportation of molecules between the nucleus and the cytoplasm

nucleoplasm

semi-solid fluid inside the nucleus that contains the chromatin and nucleolus

nuclear envelope

layer of two membranes that surrounds the nucleus of a cell

cell cycle

series of events that cells go through as they grow and divide

G1 phase

The first gap, or growth phase, of the cell cycle, consisting of the portion of interphase before DNA synthesis begins.

S phase

Replication is restricted to S phase to ensure that the genome (total DNA) is duplicated once and only once per cell cycle before cell division, preventing genome instability

G2 phase

The second growth phase of the cell cycle, consisting of the portion of interphase after DNA synthesis occurs.

Humans are diploid

Each somatic cell contains two copies of the chromosome

One from each parent

gametes

Eggs and sperm are haploid cells, meaning they contain half the number of chromosomes of the organism. In humans, each egg and each sperm contains23 chromosomes

zygote

When an egg and a sperm fuse during fertilization, the resulting cell becomes diploid, containing the full number of chromosomes for the organism —has 46 chromosomes and is diploid

Human Haploid genome

(one set (23) of chromosomes): ≈ 3.2 billion nucleotides in the23 chromosomes

Human Diploid somatic cell

(two sets (46) chromosomes): ≈ 6.4 billion nucleotides in the46 chromosomes

21st chromosome

Only chromosome can be duplicated (mutation) and still produce viable human life (other than sex chromosomes)

Origin of replication (ORI)

Each mammalian chromosome is composed of many replication sites

Multiple origins allow eukaryotes to replicate their larger DNA in a relatively short time

Replication must be fast and accurate --> cell cycle

telomeres

Repeated DNA sequences at the ends of eukaryotic chromosomes

DNA polymerase

The enzymes which synthesize or polymerize the new DNA strand during DNA replication

How DNA polymerase works

DNA polymerase uses energy stored in triphosphate of the incoming nucleotide to form the phosphodiester bone and extend DNA strand

pyrophosphate

byproduct of DNA synthesis which hydrolyze to form 2 Pi; makes the reaction irreversible

What if a nucleoside used a diphosphate

the reaction would be closer to equilibrium and reversible

DNA Exonucleases

Enzymes which remove nucleotides DNA

specific exonucleases can remove nucleotides from the 3' end or 5' end of DNA

3' to 5' exonuclease

The repair mechanism in DNA Polymerase that cuts out DNA to repair it when there is a mismatched basepair

5' to 3' exonuclease

removes RNA primer

POLYMERIZING DNA polymerases

an only polymerize (add nucleotides to the DNA strand) in one direction, the 5′ to 3′ direction

Alpha polymerase

only one that can do de novo synthesis

RNA-DNA hybrid primer synthesis on both leading and lagging DNA strands

POL A/primase complex initiates DNA synthesis in eukaryotic replication

Beta polymerase

DNA repair

Gamma polymerase

replicate mitochondria DNA

- 3' to 5' exonuclease (proofreads)

Delta polymerase

polymerize (replicate) DNA on lagging strands

fill DNA gaps when primer is removed

- 3' to 5' exonuclease (proofreads)

Epsilon polymerase

polymerize (replicate) DNA on leading strand

- 3' to 5' exonuclease (proofreads)

POLYMERIZING DNA polymerases can only polymerize nucleotides in one direction

the 5′ to 3′ direction

DNA Chain Elongation by

occurs in 5' to3' direction by adding one nucleotide at a time to 3'-OH of the nucleotide

when a nucleotide is added

two terminal phosphates cleaved off:

provides newly exposed 3'- OH

3'-OH end

an participate in addition of another nucleotide as DNA synthesis proceeds.

DNA polymerase delta and epsilon

contain exonuclease activity 3'-5' for proofreading their polymerase activity

Exonuclease activity 3'-5'

proofread newly synthesized DNA, remove/replace incorrect nucleotides

DNA mismatch repair

Mechanism for recognizing and correcting incorrectly paired nucleotides—those that are noncomplementary.

Consensus sequence

a sequence of DNA having similar structure and function in different organisms

origin of replication

Site where the replication of a DNA molecule begins; consensus sequence; eukaryotic cells have multiple

replication bubble

pre-replication protein complex opens the two strands of DNA at the origin of replication

TATA box

crucial promoter DNA sequence; A-T bonds with only two hydrogen bonds are weaker, and easier to separate

bidirectional replication forks

When the replication bubble forms, replication proceeds as the replication forks move, unwinding double stranded DNA

Pre-replication complex

a protein complex that forms at the origin of replication during the initiation step of DNA replication

Helicase

Uses ATP as energy to break the H-bonds; After the replication bubble is created, this enzyme unzips the double-stranded DNA helix by breaking the hydrogen bonds between the bases

Nucleoside triphosphate pool

essential, tightly regulated, and dynamic building blocks for RNA/DNA synthesis

Single stranded binding protein (SSBP)

Bind the DNA strands and keep them from reannealing (prevent hydrogen bonds to form between the bases)

Why need SSBPs?

The complementary strands want to reanneal after the hydrogen bonds between them are broken

template/parental strand

a strand of DNA that is used to synthesize a complementary strand of DNA or RNA

primer

A short segment of DNA that acts as the starting point for a new strand:

eukaryotic DNA replication is a RNA-DNA hybrid

Short RNA- DNA stretch made by DNA Pol (Alpha)/primase; can start DNA synthesis from scratch

DNA Pol (Alpha)/primase

the only DNA Pol which also has a subunit with primase activity

can only polymerize in the 5' to 3' direction

- synthesizes a short RNA-DNA hybrid (10 RNA nucleotides followed by20 - 30 DNA nucleotides)

-Details poorly understood

In bacteria, the enzyme "Primase"

is a type of RNA polymerase

uses nucleoside triphosphates to make a short RNA stretch complementary to the 3' end of the template

RNA polymerases

can start new chains from scratch

RNA chains are made 5' to 3'

delta and epsilon DNA polymerase

- cannot start DNA replication from scratch, need alpha DNA polymerase to set a primer with a free hydroxyl

- use it 3'-5' exonuclease activity to proofread any mistake it makes during replication

RNA/DNA primer

primase end of alpha DNA polymerase adds 10 RNA bases in primase pocket, then the DNA polymerase end builds off that and adds 20-30 DNA bases

primase

An enzyme that joins RNA nucleotides to make the primer using the parental DNA strand as a template.

primase pocket

specialized, conserved binding sites within primase-polymerase enzymes that bind DNA templates and nucleotides, with distinct pockets used for initiation (I-site) and elongation (E-site)

How many nucleotides are added in DNA replication

α DNA pol adds 50-100 pairs total, and δ/ε extend it further by 100-10000 nucleotides

Nucleotides added to DNA chain

must be in the form of nucleoside triphosphates (dNTP)

- deoxyAdenosinetriphosphate (dATP)✓

- deoxyGuanosinetriphosphate (dGTP)✓

- deoxyCytidinetriphosphate (dCTP)✓

- deoxyThymidinetriphosphate (dTTP)✓

- Uridine triphosphate (dUTP)

energy to create the phosphodiester bonds

Hydrolysis of the nucleoside triphosphate bonds to release two phosphate groups + Energy

leading/continuous strand

Can only add nucleotides in the 5' to 3' direction

The bases must be complementary

Polymerization towards the replication fork

lagging/discontinuous strand

Can only add nucleotides in the 5' to 3' direction

The bases must be complementary

Polymerization AWAY from the replication fork

How many primers are needed for the leading strand

one, primer is placed and then DNA replication continues as DNA strands unravel

Topoisomerase

Enzyme that functions in DNA replication, helping to relieve strain in the double helix ahead of the replication fork; relieve DNA supercoiling

okazaki fragments

how lagging strand is synthesized

Small fragments of DNA produced on the lagging strand during DNA replication; primer + 100-200 DNA nucleotides

Why does the lagging strand synthesize incrementally

for efficiency; because the lagging strand is built away from the replication fork, the primer is not able to lay where the DNA is still wound

must work in fragments as DNA unwinds

proteins/enzymes used in DNA replication

work until they cant anymore/are old, from where they are sent to the degradation pathway and harvested for amino acids

ribonuclease enzyme

RNase H removes the RNA part of the primers

DNA polymerase delta's special job

adds DNA nucleotides to replace the RNA nucleotides that was removed (for both leading and lagging strand)

- cannot bind these new nucleotides with Okazaki fragments because they have monophosphate ends

DNA ligase

Catalyzes the formation of phosphodiester bonds; joins okazaki fragments

makes diphosphate on okazaki fragment end (using ATP or NAD)

result of DNA replication

two identical strands that each contain one old strand and one new strand

Each of these strands will end up in two daughter cells by the end of mitosis

homologous chromosomes

Chromosomes that have the same sequence of genes and the same structure; slightly different DNA sequences

Sister chromatids

Identical copies of a chromosome; full sets of these are created during the S subphase of interphase.

DNA replication must be error free.

Errors can cause mutation in genes which can lead to diseases

must be quick and accurate

DNA polymerase has proofreading abilities

DNA polymerase has 3' to 5' exonuclease activity

During DNA replication, is it impossible for the wrong base to be inserted?

Not impossible - biochemical process, disfavored Keq(energetically less good)

there is an equilibrium constant for inserting the wrong base

Keq

original accuracy + proofreading = 10^-6

mismatch repair

original accuracy + proofreading + mismatch = 10^-9

What happens if an error is detected?

bases around error are cut out and then beta DNA polymerase fills in correct sequence