BCH Lecture 504: DNA IV: Eukaryotes (Humans)

initiation: where to start

where to start: origin of replication (oriC)

initiation: how to access the template

DnaA DnaB DnaC

Elongation: How to read and write

DNA polymerase III (Bidirectional Replication Fork

Termination: Where to stop

tus/ter complec

Genomic structure of eukaryokes

linear chromosomes

large range of genome size amongst eukaryotes

multiple origins of replication on each chromosome

condensed via chromatin

genomic structure of humans

46 linear chromosomes

multiple origins

dna wrapped around proteins called

histones

dna+histone=

nucleosome

euchromatin

loose confirmation

heterochromatin

tight confirmation

prior to DNA replication Nucleosomes must

be removed

G0

resting phase (most differentiated cells)

G1

gap 1 phase

S

synthesis phase

g2

gap 2 phase

M

mitosis

prior to dna replication histones

need to be removed, otherwise they will block the replication machinery

DNA synthesis occurs during which phase

S phase

Regulation occurs during which phase

G1 phase

goal of initiation (g1 phase)

to load and activate the helicase (origin licensing)

Origin Recognition Complex

bind origin of replication

ORC recruits (step 1)

Cdc6 and Cdt1

Cdc6 and Cdt1 recruit (step 2)

minichromosome maintenance protein (MCM) complex

Pre initiation complex (PreRC) (step 3)

right before everythin falls off and is activated

all initiation factors besides MCM complex

dissociate from DNA and initiation is completed

Cyclin CDK complexes

regulate cell cycle

cyclin binds

substrates c

cylin regulates

progression through cell cycle

cyclin dependent kinase (CDK) phosphorylates

substrates bound by cyclin

cylin d is active

during g1

cyclin e

bridges g1 and s phase

cyclin a

active during s and g2 phase

cyclin b

bridges g2 and m phase

cuclin expression/ activity is regulated by

cyclin CDKs

To initiate DNA replication in eukaryotes

the origin recognition complex (ORC) will bind the DNA.

Cyclin-CDK complexes will phosphorylate

some proteins present at the origin of replication,

the phosphorylation of some proteins present at
the origin of replication,

causes all proteins (except the helicase) to fall off the DNA

once everything falls off the dna,

the helicase (MCM) becomes active and DNA replication will
start.

DNA Pol α (Primase and Polymerase)

No proofreading; Poor processivity (no siding clamp

DNA Pol δ

Requires sliding clamp for processivity

DNA Pol ε

Sliding clamp not required for processivity

Polymerase Switching

• Swapping one DNA polymerase for another during elongation

• Occurs on leading strand (DNA Pol δ to DNA Pol ε)

• And during Translesion synthesis (LECTURE 505)

1a.) Primase (Pol α) adds

RNA primer (ntps)

1b.) Polymerase (Pol α) takes over and

adds some dNTPs

• Poor processivity causes Pol α to dissociate

2.) dNTPs are added by

replication machinery

Polymerase δ adds

some nucleotides

polymerase switch

Swapping one DNA polymerase for another during elongation (DNA Pol δ to DNA Pol ε)

DNA Polymerase ε adds

majority of nucleotides

dna is synthesized in ___ direction

5’ to 3’

dna template is read 3’ to 5’

3’ to 5’

3 different and distinct polymerase are required to s

synthesize dna in eukaryotes

Leading strand requires

all 3 polymerases (Pol α, δ, and ε).

Pol ε does ___ and requires ___

most of the synthesis and does not require a sliding clamp

Lagging strand requires

2 polymerases (Pol α and Pol δ)

Pol δ does ___ and requires___

most of the synthesis and requires a sliding clamp (PCNA).

Primers are removed from DNA via

Pol δ and FEN1.

Termination in eukaryotes occur when:

two replisomes collide or replisome reaches end of chromosome

Termination in eukaryotes occur when

DNA polymerases collide, or if they fall off the end of the chromosome.

he primer located at the end of the genome is a major issue because

we cant leave it there but we lack the machinery to replace the primer with deoxynucleotides

so the primer is removed but

not replaced with DNA

Telomeres are made of

thousands of copies of repetitive sequences at the tips of each chromosome (acts as protective tips)

downside of telomeres

shortening of chromosome compunds

when telomoeres become too short

cells become senescent (no longer able to replicaate)

telomeres have

Non-coding, repetitive, hexameric sequences

Ribonucleoprotein

contains both protein and rna

telomerase adds

additional repeats to telomeres

TERT

protein subnuit

Telomerase reverse transcriptase

Telomeres are added to

the ends of linear chromosomes by

telomerase.

Telomerase consists of

protein and rna components

telomeres prevent the loss of

functional dna from thr ends of chromosomes but do not prevent the loss of telomere DNA

in stem and stem like cells telomerase is

active

telomerase is ___ in most cells besides stem cells

inactive