[‼️‼️‼️‼️] MASTER MASTER MASTER BIOL 300 STUDY CARDS

UNLV

★ Why is one round of replication per cell cycle important?

Uncoupling causes gene loss (too few replications) or polyploidy (too many, endocycle)

Default state of prokaryotes vs multicellular eukaryotes?

Prokaryotes divide by default; eukaryotes do not divide by default

Where is the eukaryotic G0 state?

Quiescent cells, exit from the cycle before G1

What two things must a cell be to re-enter the cycle from G0?

Competent (responds to signals) and in good shape (passes checkpoints)

Three main eukaryotic cell cycle control inputs?

Growth factor signaling (promote), checkpoints (allow), cyclin/CDK levels and activity

What kinds of ligands and receptors start growth factor signaling?

Growth factors bind receptor tyrosine kinases

Primary effector of growth factor signaling?

Ras (activated by a Ras GEF)

★ What is Ras's active form?

Ras-GTP (binds downstream effectors)

What is Ras-GDP?

Inactive form, cannot bind effectors

Which proteins switch Ras on and off?

GEF activates (GDP to GTP), GAP inactivates (GTP to GDP)

Why is Ras an oncogene?

Mutations lock it in GTP form (cannot hydrolyze GTP or bind GAP), so it stays on

How do cyclins and CDKs work?

Cyclin binding activates CDK; cyclin levels oscillate, CDK protein levels stay constant

★ Three ways CDK activity is controlled?

Cyclin binding, phosphorylation (CAK activates, inhibitory kinases and cdc25 phosphatase), CKI inhibitors (p21, p27)

Is cyclin binding enough to fully activate CDK?

No, necessary but not sufficient

What do G1-S checkpoints check?

Cell size (enough growth) and DNA damage (repaired)

Why is replicating damaged DNA the worst outcome?

Causes death or tumorigenesis

★ What is a tumor suppressor?

Gene that inhibits the cell cycle; loss of function causes tumors

What is an oncogene?

Mutant allele that drives the cycle without signals (often hypermorph, e.g., constitutively active Ras)

★ Two key checkpoint genes?

Rb and p53 (both transcription factors); their LOF is the most common tumor suppressor loss

★ What does p53 do after DNA damage?

Stabilizes, then either repairs (if damage is repairable), arrests, or triggers apoptosis (if catastrophic)

What is Rb's role?

Integrates growth factor, damage, and size signals; controls the restriction point

★ How does Rb block the cycle?

Binds E2F transcription factors and keeps them from turning on S-phase genes

How does Rb release E2F?

Phosphorylation by cyclin/CDK releases E2F

Why is Rb the first tumor suppressor identified?

Its LOF causes most retinoblastoma

Retinoblastoma: why is one eye more often affected in non-hereditary cases?

Needs two hits in one retinal cell lineage; a single hit is one mutagenesis event

Heritable vs non-heritable retinoblastoma?

Heritable: born heterozygous, earlier onset, both eyes. Non-heritable: later onset, usually one eye

What is inherited in hereditary retinoblastoma?

The susceptibility, not the disease itself

★ What is an origin of replication (ori)?

Specific DNA sequence where replication starts; AT-rich; proteins bind it specifically

★ What is a replicon?

A DNA piece controlled by one origin

Origins in prokaryotes, phages, plasmids, organelles?

A single ori

Origins in eukaryotic chromosomes?

Tens of thousands, spaced about 40 to 100 kb apart

★ Why do eukaryotes need many origins?

Their genomes are much larger; one origin per replicon keeps the whole genome replicable in time

Why not fire all origins at once?

Simultaneous firing would take about 1 hr; real S phase takes about 6 hr, so firing is coordinated

Semiconservative replication: what is conserved?

Each new duplex has one old and one new strand

What did Meselson and Stahl show?

DNA replicates semiconservatively (heavy nitrogen labeling)

★ Unidirectional vs bidirectional replication?

Unidirectional: one fork per origin. Bidirectional: two forks moving opposite directions per origin (most common)

Do unidirectional and bidirectional bubbles look different?

No

★ What is oriC?

E. coli origin, about 245 bp

★ What are DnaA boxes?

DNA sequences that bind DnaA, the replication initiator

★ What are the 11 GATC repeats in oriC for?

Adenine methylation on both strands, required for DnaA binding

What does DnaA-ATP do?

Binds origin repeats, forms an oligomer that melts DNA

★ Why is DnaA a licensing factor?

It is active only when bound to ATP and only when replication is needed

How is DnaA inactivated (prevents re-initiation)?

Its intrinsic ATPase converts DnaA-ATP to DnaA-ADP; stimulated by membrane lipids and ssDNA

Why is oriC attached to the membrane?

Membrane lipids stimulate DnaA ATPase, and sequestration blocks methylation

★ What is the DnaB helicase?

Hexameric helicase that unwinds DNA at the fork; loaded onto origin by DnaC

What does DnaG primase do?

Makes RNA primers for DNA synthesis; bound to the helicase

What does the DNA unwinding element (A-T rich) do?

Easier to melt, so the initial strand separation starts there

Role of gyrase and SSB at the fork?

Gyrase relaxes supercoils ahead; SSB coats ssDNA to keep it from reannealing

★ Two functions of methylation in bacterial replication?

Required for DnaA binding (initiation); marks the old strand for mismatch repair

Why is hemimethylated DNA not replicated?

Newly synthesized DNA is hemimethylated and cannot initiate, so premature replication is prevented

Two ways replication is prevented from starting again in bacteria?

DnaA-ATP to ADP conversion, and methylation delay (oriC sequestered at membrane)

★ Cis-acting vs trans-acting?

Cis: DNA element acted on (origin). Trans: protein expressed from elsewhere that binds it

Bacterial cis and trans pair?

oriC (cis) and DnaA (trans)

Yeast cis and trans pair?

ARS (cis) and ORC (trans)

★ What are ARS elements?

AT-rich autonomously replicating sequences, including elements A, B1, B2, B3

Which ARS element is essential?

Element A; A plus any two B elements is sufficient

★ What is ORC?

Origin recognition complex; multi-subunit DNA-binding complex, conserved in all eukaryotes

When does ORC bind B2 and B3?

Only when it is time to replicate; it contacts A and B1 all cycle

★ What is licensing in eukaryotes?

In G1, licensing factors load inactive MCM helicase onto origins to form the pre-RC

★ What is firing?

G1 to S, initiation factors activate MCM and recruit replication machinery, converting pre-RC to pre-IC

What is post-RC?

After replication, licensing factors are degraded or absent, so origins can't refire until the next cycle

★ How do eukaryotes prevent premature re-initiation?

Degrading licensing factors (vs bacteria: methylation of oriC)

Why do replicons near active genes replicate early?

Early replicating regions are near active genes; heterochromatin replicates late

How many replication forks are active at once in eukaryotic S phase?

Only about 15% of origins fire at the same time (per slide)

What is quick-stop mutant?

Growth stops immediately upon temperature shift; mutated protein is in elongation

★ What is slow-stop mutant?

Growth stops slowly; mutated protein is involved only in initiation

What are temperature-sensitive (conditional lethal) mutants?

Functional at permissive temperature, lose conformation and function at restrictive temperature

Why does a slow-stop mutant indicate initiation defect?

The cell can finish current forks, then no new ones start

Three classes of DNA polymerases in cells?

Replicases (genome replication), repair polymerases, translesion (error-prone) polymerases

Why are different DNA polymerases needed?

Different types of DNA synthesis (replication, repair, bypass, organelle) need different fidelity and processivity

★ What does every DNA polymerase do?

Forms a phosphodiester bond between 3'-OH of the chain and 5'-phosphate of the next nucleotide

★ Synthesis direction?

5' to 3' only

★ What does DNA polymerase need to start?

A primer with a free 3'-OH (provided by RNA primase)

Why does telomerase exist?

To solve the end replication problem (no primer at the 5' end)

Shape of polymerase active site?

Palm (catalysis), fingers (positions template), thumb (grips DNA for processivity)

★ What is proofreading?

3' to 5' exonuclease activity that removes mismatched bases

★ Two sources of replication errors?

Substitutions (base mismatch) and frameshifts (slippage)

How does polymerase prevent substitutions?

Tight active site fits only Watson-Crick pairs; proofreading removes errors that get through

Why are error-prone polymerases error prone?

Open active site fits non-Watson-Crick pairs

★ What causes frameshifts?

Polymerase dissociates and reassociates; most likely on homopolymeric runs

What determines frameshift fidelity?

Processivity: tendency to stay on the same template

★ Helicase function?

Separates strands using ATP (about 1 ATP per bp), hexamer, DnaB in bacteria

Function of SSB?

Binds single-stranded DNA so it cannot re-form duplex

★ Primase function?

Synthesizes short RNA primers (about 11 to 12 nt)

Length of bacterial and eukaryotic Okazaki fragments?

Bacteria about 1 to 2 kb; eukaryotes about 100 to 200 bp

★ Leading vs lagging strand?

Leading is continuous; lagging is made as Okazaki fragments (discontinuous)

Why is replication "semidiscontinuous"?

One strand is continuous, the other is discontinuous

How is the RNA primer on Okazaki fragments removed?

DNA Pol I removes RNA and fills the gap

★ What seals the nick between Okazaki fragments?

DNA ligase

★ What makes up Pol III holoenzyme?

About 10 proteins (900 kD): two catalytic cores, two clamps (beta), one clamp loader (gamma)

What is the beta clamp?

Homodimer that holds the core on DNA for processivity

What does the clamp loader do?

Places and releases beta clamps on DNA using ATP

Dimerizing subunit of Pol III?

Tau links two cores, one for each template strand

Why is replication energy intensive?

Helicase, gyrase, primase, and Pol III each need ATP

Why does a linear DNA end need telomeres?

Bare ends are read as double strand breaks and get joined (sticky ends)

★ What is the end replication problem?

Lagging strand cannot be fully primed at the 5' end, so DNA shortens each round

★ How does a T-loop protect the end?

The 3' overhang loops back and displaces the complementary duplex

★ What is telomerase?

RNA-dependent DNA polymerase (ribonucleoprotein); its RNA template extends the 3' end

What are telomeres made of?

Long tandem repeats (100 to 1000 copies)

Are telomeres like centromeres?

Telomere repeat sequence matters for its structure and function