BIOL 300 Exam II

Chromosome Separation (Bacteria Division)

• Topoisomerase

• Recombination

Septum Formation (Bacteria Division)

Same as bacteria envelope

Rod shape is important

Minicells (Division Mutants)

Misplaced septum

Genes involved in septum positioning

Multinucleated filament (Division Mutants)

Failure in septum formation

Genes involved in septum formation

Anucleate Cells (Division Mutants)

Failure in chromosome separation

Genes involved in chromosome separation

Temperature Sensitive Allele

Mutant protein unfolds and loose normal structure at high temp

Wild-type at normal temp, but mutant at high or low

Growth Factor Signaling Ligand

Growth factors

Growth Factor Signaling Receptors

Receptor Tyrosine Kinase (DN allele)

Growth Factor Signaling Primary Effector

Ras GEF

Growth Factor Signaling Second Messenger

Ras-GTP (CA allele, oncogene)

Growth Factor Signaling Effectors of Ras

A cascade of kinases

Ras-GDP/GTP Switch

Ras: Small GTPase (enzyme that hydrolyze GTP)

Signaling activity is to bind and activate downstream kinases (2nd messenger)

GTP binding → active → bind downstream

GDP binding → inactive → cannot bind

Constitutively Active Alleles

Hypermorph

Mutations in GF receptors w/o GFs

Oncogene

Mutant alleles that promote tumorigenesis

CDK

Cyclin-Dependent Kinases

CDK Regulation

Protein levels remain largely constant throughout cell cycle

Their activities oscillate during cell cycle

Cyclin Binding

Releases CDK self-inhibition

Cyclin protein levels are cyclical in cell cycle

Necessary but not sufficient to fully activate CDK

Phosphorylation

CAKs (CDK activation kinases), inhibitory kinases, and phosphatase

CKI

CDK inhibitor

Block cyclin binding

p27/21

Cell Cycle Checkpoints

At G1-S transition, they check for sufficient cell size growth and DNA damage

Promote cell cycle arrest if mechanism senses defects

Rb and p53

Tumor Suppressors

Genes that function to inhibit cell cycle

LOF leads to tumorigenesis

“Guardians” of cell integrity

Rb

Retinoblastoma protein

First tumor suppressor identified

LOF in Rb is responsible for most cases of retinoblastoma

Upstream: Integrates GF signaling, DNA damage, and cell size pathways

Downstream: Binding and inhibiting E2F transcription factors that turn on genes required for passing G1 and entering S

Retinoblastoma

LOF mutations in Rb are recessive

Need both alleles to be mutated in order to develop tumor (homozygous)

Origin of Replication (ORI)

Type of DNA elements = DNA sequences that have specific functions (almost always means that certain proteins can specifically bind to that sequence)

Replicon

A piece of DNA controlled by the same one origin

Cis Element

“Origin”

The DNA

Trans Factor

Origin binding proteins and DNA polymerase

The protein

Autonomous Replicating Sequence (ARS)

AT rich domains (B3, B2, B1, A)

Element A contains a consensus sequence and is essential

A plus any two B is sufficient

Origin Recognition Complex (ORC)

Multi-subunit DNA binding complex that recognizes the ARS

Highly conserved

ORC contacts A and B1 throughout most of the cell cycle

ORC binds to B2 and B3 only when it’s time to replicate

Licensing

During G1, licensing factors recruit inactive MC helicase to the origins, forming pre-replication complex (Pre-RC)

Licensing factors are unstable, and their degradation (absence) blocks replication until next cell cycle

Firing

At G1 to S, initiating factors activate MCM and recruit replication machinery, converting Pre-RC to pre-initation complex (Pre-IC)

Quick-Stop Mutants

Cell growth stops immediately upon temperature shifts

Mutations in components of replication machinery = mutations in proteins involved in elongation

Slow-Stop Mutants

Cel growth stops slowly

Mutations in proteins exclusively involved in initiation

DNA Polymerases Activity

Catalyzing the formation of phosphodiester bonds between 3’-OH of one nucleotide and 5’-triphosphate of the next nucleotide

Requires a primer (existing 3’-OH from a chain) → primer is RNA

5’ → 3’ direction

Palm

Catalytic active site

Fingers

Positions template in the active site

Thumb

Binds DNA as it exits enzyme

DNA Polymerase Proofreading

E.Coli Pol I

3’ → 5’ exonuclease activity

Fidelity

The ability of the polymerase to avoid or to correct errors in the newly synthesized DNA strand

Substitution

Determined by the efficiency of base-pair discrimination (shape of catalytic center) and proofreading (3’ → 5’ nuclease activity)

Frameshift/Replication Slippage

Determined by the processivity of the polymerase (tendency to remain on a single template rather than to dissociate and reassociate)

Preventing Substitution

Catalytic center only llows Watson-Crick pairs to form

Proofreading allows wrong base-pairs to be removed

Preventing Frame Shift

Determined by the processivity of the polymerase (finger and thumb)

Replication slippage can only occur when polymerase dissociates and reassociates

Tend to occur during the replication of a homopolymeric stretch

Replication Steps

1. Separation of DNA strand: DNA Helicase (DnaB)

2. Single-strand DNA protection: Single-strand binding protein (SSB)

3. Primer synthesis: Primase (DnaG)

4. DNA Synthesis: DNA Pol III holoenzyme

5. Joining Okazaki fragments: Pol-I and ligase

Separation of DNA strand

Uses energy from ATP hydrolysis, 1 ATP = melt 1 bp

Typically hexamer with 6 binding sites for DNA

Single-Strand DNA Protection

Binds ssDNA at replication fork; keeps them from reforming duplex

Binds as monomer, but the addition of more subunits is cooperative

Primer Synthesis

Special RNA Pol that only synthesizes short stretches of RNA (11-12 nts)

Leading Strand

Continuous w/in each replication

Lagging Strand

Not continuous w/in each replication

Okazaki fragments

Dipolymerase Model

Explains how leading and lagging strands are synthesized at the same time

E.Coli Pol III Subcomplex Components

Catalytic core (2x)

Dimerizing subunit

Clamp (2x)

Clamp loader

Catalytic Core

α subunit = pol activity

ω subunit = 3’ - 5’ proofreading exonuclease

θ subunit = stimulates the exonuclease

Dimerizing Subunit

2x t subunit = links the two cores, one for each template strand

Clamp

Holds catalytic core on DNA template

Each clamp is a homodimer of β subunits that binds around DNA and ensures processivity (prevents Pol core from dissociating from template)

Clamp Loader

y complex consisting of 7 proteins from 5 genes (c, y, d, d’, g)

Needed to place and release the clamp on DNA

Energy consuming (use ATP)

Joining Okazaki Fragments

1. Removal of RNA primer: DNA Pol I (repair polymerase)

2. Nick ligation: DNA ligase makes bond that connects 3’ end of one fragment to 5’ end of another

Role of ATP at the Replication Fork

1. For helicase (DnaB): to unwind duplex

2. For gyrase (type of topoisomerase): needed for relaxing, swiveling action

3. For primase (DnaG): to start primer

4. For DNA pol III: requires it for action

Homologous Recombination

Depends on the sequence homology between two DNAs

Between any long enough homologous sequences

Site Specific Recombination

Use an enzyme that recognizes the short sequence

Often results in deletion/excision

Between a pair of very short homologous sequence

Somatic Recombination

W/in the same chromosome

Results in deletion

Double-Strand Break Recombination (DSBR)

Meiosis I (spematogenesis/oogenesis)

Two DNA double helices

Crossover

DSBR between two homologous chromosomes at the equivalent locus do not lead to new mutation

Non-sister chromatids (maternal/paternal)

Synthesis-Dependent Strand-Annealing (SDSA)

Interphase (can be interphase of mitosis)

Two DNA double helices

Non crossover

Single-Strand Annealing Model (SSA)

One DNA helix

Roles of Homologous Recombination

Contribute to genetic diversity through allele shuffling

A mechanism of precise repairs of DNA double strand break

Double Strand Break Repair Mechanism Step 1

DSB is actively produced by endonuclease called Spo11 in the “recipient” duplex

Ends of DSBs are covalently bound to Spo11 to protect them

Double Strand Break Repair Mechanism Step 2

5’ end Resection

Exonucleases and endonuclease digest 5’ end of the break

Creates a 3’ free end

Double Strand Break Repair Mechanism Step 3

Homology searching and invasion

One free 3’-end invades a homologous region in the '“donor” duplex

Creates a region of heteroduplex and the D-loop

Double Strand Break Repair Mechanism Step 4

Extension of the invading strand

DNA synthesis

Recipient strand is elongated using the donor duplex as the template

The recombinant joint can move along the duplex → called branch migration

Double Strand Break Repair Mechanism Step 5

Second strand invasion/capture

The second resected end in the recipient strand anneals to the donor strand

Recipient strand is extended to complete the second recipient strand

D-loop is converted into two Holliday Junction

Double Strand Break Repair Mechanism Step 6

Resolution of Holliday junctions

If both junctions “cut” in the same way (both vertical/both horizontal) → noncrossover DNA

If two junctions are resolved in the opposite ways → crossover recombinant DNA

Synthesis-Dependent Strand-Annealing Mechanism Steps 1-4

Same as DSBR

Synthesis-Dependent Strand-Annealing Mechanism Step 5

After the DNA synthesis, the invading strand goes back

No crossover

Synthesis-Dependent Strand-Annealing Mechanism Step 6

Synthesis of the other strand

Ligation

SDSA Mechanism Differences

No second invasion

No Holliday junction

No crossover product

Single-Strand Annealing Model

Occurs at DSBs between direct repeats (same direction)

Within one DNA/not between two DNA

Results in LOSS of DNA information: sequences between the repeats

Heteroduplex DNA

Region where strand from non-sister chromatids anneal to each other

Mismatches will be repaired using the mismatch repair systems

If allele sequence changes → “Gene Conversion”

Chi Site

Occurs naturally in E.Coli abot once every 5-10 kb

Stimulates recombination in its general vicinity creating hotspots

RecBCD Complex

Avidly binds to DSB

Multiply enzymatic activities (Helicase → RecB on 3’, RecD on 5’; Nuclease → RecB)

Recognition of Chi cite by RecC reslts in many changes in enzymatic activities, leading to generational ss 3’ end and RecA recruitment

RecA

Coating ss 3’ end and catalyze 3’ strand invasion

Polymerizes along either ss or ds DNA to form a nucleofilament

Promote base paring between a ss of DNA and its complement in a dplex molecle

Homologs exist in every kingdom of living systems

Three Condition 3’ SS Invasion

SS region and the 3’ end must be located w/in a region that is complementary between the molecule

One of the DNA must have a ss region

One of the DNA must have a free 3’ end

Holliday Junction Formation Following Strand Invasion

Reaction catalyzed by RecA between two duplex molecles leads to the formation of a Holliday junction

5’ resection produces the free 3’ end and the ss

Ruv Genes

Repair of UV

Recombination is needed in the repair of DNA DSBs, therefore recombination mutants are often sensitive to agents that cause DSB

UV irradiation → DNA DSBs → damages not repaired in time → DSB accumulate to high levels → slow grow or death

RuvA

Recognizes Holliday structure and binds to all 4 strands

RuvB

Hexamer ATP dependent helicase for branch migration (10-20 bp/s)

RuvC

Endonuclease that specifically recognize and cleaves Holliday junctions

End Processing (Bacteria)

Nucleases

RecBCD, Chi site

Invasion and Holliday Junction Formation (Bacteria)

RecA

Branch Migration (Bacteria)

Helicase

RuvA&B

HJ Resolution (Bacteria)

Endonuclease

RuvC

Integration (Phage λ)

One DNA is circlar

Catalyzed by integrase

Excision (Phage λ)

Two elements on the same DNA deletion (reverse of integration) catalyzed by different enzymes

Site-Specific Recombination Differences

Short recognition seqences

Enzyme recognizes short sequence: HR occurs throughout genome

No double-break → sequential nick cuts

No RecA-mediated invasion

No DNA digestion and synthesis involved

No need for additional energy

Only one Holliday junction (always crossover)

Germline Mutations

Mutations in sperm and egg DNA or their progenitors

Can be inherited by offspring (whole body)

Drive evolution → subjected to natural selection or genetic drift

Somatic Mutation

Cannot transmit to progeny

Could lead to cancer if a mutation turns proto-oncogenes to oncogene or lead to LOF of tumor supressors

Single-Base Changes

Do not grossly distort overall structure

Can be detected by repair systems but does not block transcription or replication

Caused by replication errors (mismatched base pairs) and base conversions

Structural Distortion

Distortion enough to physically impede replication or transcription

Covalent bonds formed between bases → UV irradiation leading to pyrimidine dimers

Addition of bulky adducts → distorts structure

Depurination → A or G base part lost

DNA Strand Breaks

Single strand → directly repaired by ligation

DSB → major DNA damages that, if unrepaired, lead to extensive DNA loss

Direct Reveral

No DNA synthesis

Excision (DNA Repair Systems)

Remove and re-synthesis using the complementary strand as a template

Base excision

Nucleotide excision

Mismatch excision

Recombination Repair

Remove and re-synthesis using the homologous chromosome as a template