Genes 2 Proteins DNA replication

DnaB (pro)

helicase

DnaC (pro)

brings in the helicase

DnaG (pro)

primase; makes the primer

DnaA (pro)

binds to OriC & opens up the replication bubble

What happens to DnaA when it binds to ATP? (pro)

undergoes conformational change

Replisome (pro)

2 copies of pol III, sliding clamps + loader (replication complex)

Single-strand binding protein (SSBs)

prevent DNA from forming secondary structure when separated in prokaryotes

Sliding clamp (pro)

brings in the DNA polymerase; recharges with ATP to repeat the cycle

Ligase

seals the nick in DNA

Bacterial helicase moves along the __

lagging strand

Eukaryotic helicase moves along the __

leading strand

RIDA regulation

DnaA-ATP in prokaryotes are hydrolyzed to DnaA-ADP; therefore blocking replication initiation

SeqA

competes with dam methylase to fully methylate DNA strands and delays replication initiation

DnaA binds to the __ to prevent initiation in prokaryotes

datA region

Helicases in eukaryotes & prokaryotes are both __

AAA+ proteins

Cdt1

brings the helicase in eukaryotes

Helicase (euk)

MCM2-7

PCNA (euk)

sliding clamp

__ does replication on lagging strand in eukaryotes

pol delta

__ does replication on leading strand in eukaryotes

pol E

pol-α-primase-complex (euk)

makes primer

RPA

prevents DNA from forming secondary structures

Fen1

endonuclease that cleaves flap DNA in eukaryotes

Ctf4

couples CMG helicase, pol E & pol-α-primase at replication fork

Pol III (pro)

synthesizes 5’ → 3’; disassociates when it reaches RNA primer

Pol I (pro)

removes primer and repairs gap

t complex

links the polymerases & associates with the clamp loader and helicase

Active site in polymerase

catalyzes a phosphoryl transfer reaction that links the 5’ end phosphate of the incoming nucleotide to the 3’ end of the growing DNA

What do the 2 magnesium ions at the active site do?

one activates the 3’ OH & the other interacts with the incoming nucleotide and stabilizes the negative leaving oxygen

What is the active site made up of?

2 aspartate residues & 2 Mg²⁺ ions

Reverse transcriptase:

DNA polymerases that copy RNA into DNA

ORC:

• eukaryotic initiator

• recruits Cdc6 & Cdt1

OriC

• prokaryotic origin sequence

• 245 bp with 7 9-bp DnaA boxes

How does the ORC bind to DNA in most eukaryotes?

sequence-independent manner

Cdc6 & Cdt1 (euk):

• recruit MCM2-7 hexamers head to head

What happens to ORC once MCM2-7 are loaded?

it dissociates

MCM2-7 pair is activated by __

accessory proteins

CMG:

• full helicase complex (polymerase, proteins)

• activated by phosphorylation to unwind DNA and start replication

Eukaryotic helicase moves __

3’ → 5’ on leading strand

Prokaryotic helicase moves __

5’ → 3’ on lagging stand

The features of pro and euk helicase suggest what?

that they evolved independently

How does DNA prevent DNA replication on different sequences

form 2ndary structures that block polymerase from copying (SSB in pro; RPA in euk)

Prokaryotic termination:

• resolving torsional stress when 2 replication forks approach one another

• meeting of replication forks and complete DNA strands

• dissociation of replisome from the DNA

__ resolves the overwinding by breaking DNA and allowing supercoils to relax

toposiomerases

__ break one DNA strand and do not require ATP

type I topoisomerases

__ break both DNA strands and do require ATP

type II topoisomerases_

__ topoisomerases act by strand passage

__ topoisomerases cleave and pass the other strand through the break

type IA

__ topoisomerases allow the free end to swivel to release supercoils before relegation

type IB

Replication forks meet in a termination zone that has __

10 ter sites

ter sites bound by the Tus protein are __

orientation specific

How does bacteria terminate DNA replication?

the opposing forks cause flap displacement and are filled similar to Okazaki fragment maturation

In eukaryotes termination occurs at __

multiple sites

What happens when euk replisomes meet?

• CMG complexes move past each other on the leading strand

• flap displacement of newly synthesized DNA

• flaps are resolved

In euk, when the double-stranded DNA is entwined, this is resolved by __

topoisomerase II

__ blocks initiation replication in prokaryotes

RIDA

RIDA function:

• after initiation, Hda stimulates hydrolysis of DnaA-ATP to ADP

• cannot reinitiate origin firing

Dam methylase function:

• pro

• methylates A in GATC sites in oriC

• SeqA binds to hemimethylated GATC and blocks binding of methylase

• access to DnaA blocked

• temporary

DnaA titration:

• pro

• after replication 2 datA copies sequester free DnaA

• after segragation, 1 copy of datA (more free DnaA)

Nucleotide exchange:

• Dna-ATP must be regeneration from DnaA-ADP

• DARS stimulate ADP → ATP exchange on DnaA

How does origin lincensing work in eukaryotes?

• origins are selected in G1

• helicases are loaded in a 2 step process

• in S phase, helicases activate to initiate origin unwinding

• origins cannot be reused until next G1

What happens when replication forks stall in euk?

DNA damage response is triggered and mitosis is arrested until errors are fixed

The end replication problem:

• only in euk

• RNA primer removal leaves gap that cannot be filled

• replication fork dissociates once leading strand finishes before lagging str

Many eukaryotes use __ to solve the end-replication issue

telomeres

Telomerase function:

• adds telomere DNA sequences into chromosome ends

• net elongation of the telomere sequence and counterbalances loss due to incomplete end-replication

Factors that set telomere length equilibrium:

• number of telomeres elongated in a cell cycle

• how many repeats are added in a single binding event

__ have active telomerase that maintains their telomeres and allows indefinite growth

> 90% of human tumors

What happens when telomeres become critically short?

chromosome ends are less protected, triggering cell cycle arrest/cell death