Genetics Exam 1 Slide questions

How to nucleotides base pair?

via hydrogen bonds

2 between A-T

3 between C-G

How do DNA strands form?

phosphodiester bonds between 5’ phosphate and 3’ hydroxyl

important features of DNA

minor groove

major groove - more area for activity, interactions

3’ hydroxyl group, 5’ phosphate group

What makes up a nucleoside?

base and sugar

dNTP = deoxyribose nucleotide triphosphate

major and minor groove

major groove is deeper, area for more proteins to interact

minor groove is shallow, less information

base stacking

provides chemical stability to DNA double helix

hydrophobic core

What form of DNA is predominant in vivo?

form B

good in high humidity and low salt environment

UV light and base stacking

decreases the ability of DNA bases to absorb UV light

reversible strand separation

can denature and renature via heat, chemicals

important for DNA replication and possible medication delivery fxns

functions of RNA

transcription, transcriptional control, translation, post-translation modifications

hereditary material for most viruses, replicate through host cell

RNA interactions

form complementary base pairs with nucleic acids

or interact with proteins (RNP)

RNA primary structure

phosophodiester backbone linkages

ribose sugar as 3’ and 2’ OH group

U H bonds with A instead of T

common secondary structure motifs

hairpin loops, double helix with minor groove binding site

noncanonical base pairs, base triples, GU wobble widen major groove site

Tertiary structure of RNA

pseudoknot motif via coaxial stacking, tetraloop motif stabilized by base pair stacking

folding times

larger molecules take longer to fold

RNA enzyme

ribozyme, self-splicing = change

amino acid properties

amine group, carbonyl group, R side chain determines properties

protein structures

1’ = sequence of aa via peptide bonds

2’ = alpha helix or beta pleated sheet via H bonds

3’ = interactions between different parts of protein chain

4’ = interactions between different protein subunits

Where would hydrophobic AA be located

on the inside of the protein

central dogma

DNA → translation → mRNA → translation → protein → modification/regulation

Regulating protein activity

allosteric regulation and phosphorylation

phosphorylation

kinase adds P group, phosphatase removes P

causes shape change, formation or dissociation of multiprotein complex

allosteric regulation

molecular binds to allosteric (not active site) cause shape change, induce or inhibit reaction/ functionality of protein

IDPs and IDRs

proteins/region of protein without well defined 3’ structure

fuzzy binding, conformational flexibility with multiple binding sites

help assemble membrane-less organelles

problems with IDRs, IDPS

prone to misfolding, aggregation

prion

protein that adopts altered conformation, self propagates

disease causing

How does DNA fit into the nucleus?

it is packaged into chromatin (DNA + histone proteins)

DNA is negatively charged, histones are + charged

Highly conserved histones that make up core octamer

H2A, H2B, H3, H4

form dimer and tetramers respectively with exposed N-terminus tails (site for regulation of histones)

Linker histones

H1, H5, H1’

link the core octamers, slightly larger, + charged

What makes up a nucleosome?

linker histones and core octamer (beads on string motif)

Euchromatin

less packaged, more accessible packaging

associated with actively transcribed genes

Heterochromatin

more packaged, less accessible, less likely to be transcription site

How do fully condensed metaphase chromosomes form?

condensin 1 and 2 multi-complex loop, promote DNA interaction and shape chromosomes

histones aid in compaction and protection

Genome organization in bacteria + archaea (prokaryotes)

small genomes < 20Mb

Genome organization in eurkaryotes

there are > 100Mb interspersed with introns, exons, and intergenic regions

The human genome

~ 40% of of Mb are genes/gene related seuqnces

only about 2% code for functional proteins

Two domain tree

LUCA (branch off of bacteria) and LECA (branch off of eurkaryotes and archaea)

Endosimbiotic theory for mitochrondria + chloroplast

archaea engulfed bacteria, origin of energy producing organelles

Archaeal genome

circular

mtDNA

mitochondrial DNA is inherited independently of nuclear genome

• strictly inherited from egg-producing parents

• mutations/inheritance can give rise to genetic diseases

• impact high-energy organs

heteroplasmy and mitrochondrial disease

leads to differences in severity + kinds of symptoms

all of mtDNA cells are identical

more enriched mutant mtDNA = higher risk of LHON disease

Bacterial genome

single, circular DNA molecule

condensed by proteins in nucleoid

not enclosed membrane, more like a compartment

Virus genome

can be double stranded linear, single stranded circular, double stranded circular

use host cell to encode their own proteins

DNA replication is

semiconservative and semidiscontinuous

occurs in 5’ to 3’ direction

created by DNA polymerase

DNA polymerase needs

an RNA primer (primase) to being replication)

the RNA primer has a 3’ OH group

Origin or replication + fork

origin = where H bonds begin being split, where DNA synthesis begins

replication fork is bidirectional, strand is split simultaneously in both directions

DNA synthesis steps

RNA primer lays down primer

Okazaki fragments are sealed by ligase on the lagging strand (new primer needed because synthesized in small parts) (5’ → 3’)

leading strand synthesis is continuous

DNA synthesis in bacteria

there is a single-well defined origin of replication

bidirectional fork makes theta structure

Human origins of replication tend to be in

multiple in each sequence

AT rich sequences (2 H bonds to break instead of 3)

replication forks are clustered in replication factories

When does DNA replication occur in cell cycle?

during the S phase

replication is regulated by CDKs

Steps before DNA replication

nucelosomes unravel dna via histone acetylation + chromatin remodeling

Origin Recognition Complex (ORC) loads helicase

helicase opens double helix by breaking H bonds

How does DNA not tangle and get messy near origin?

Single strand binding proteins - prevent DNA from rebinding

topoisomerase - changes DNA shape to relax supercoils

partial denaturation also relieves twisting strain, more likely for origin to start here (easier to get DNA apart)

PCNA

sliding clamp with protein partners

keeps DNA polymerase from falling off, helps enzymes switch

RFC

clamp loader, makes spiral that matches minor groove of DNA

Polymerase + proofreading

geometry inhibits the creation of incorrect bp

exonuclease cuts off mismatch

Exonuclease vs. Endonuclease

exo - cuts off terminal nucleotide

endo - cleaves bond in middle of strand

Protect the ends of chromosomes with telomeres and shelterin proteins

prevent info from being lost from lagging strand

Telomerase

RNP with reverse transcriptase activity

binds to repeats on lagging strand overhang and acts a template for more repeats to extend the telomere

Regulating telomerase

t-loop and shelertin to block access

only recruited in S-phase, leaves from Cajal bodies in nucleus, find DNA via random diffusion/exploration

Hayflick limit

the point at which cells stop dividing and begin cellular aging (senescence)

some cells that aren’t rapidly dividing have low telomerase activity, chromosomes shorten progressively

too much or too little telomerase activity

too little = cellular senescence, aging

over activation = immortal, cancer cells

Spontaneous mutations

result from natural processes in cells like DNA replication errors

point mutation

a nucleotide is substituted for the wrong nucleotide

happens if DNA pol fails to proofread

Will a nucleotide substitution have a phenotypic effect?

dependent on the region of DNA

is it in a gene regulatory region? is it in an area that codes for a functional RNA molecule

Silent mutation

change one bp without changing phenotype

Missense mutation

change one nucleotide and change the protein that is coded

Nonsense mutation

change in nucleotide causes a stop codon, no more mRNA can be translated

nearly always makes nonfunctional product

Indels cause

insertions and deletions cause frameshift mutations

Trinucleotide repeats

can result in slippage intermediate which expands the trinucleotide repeat, adding extra codons

Induced mutations

due to DNA damage from outside agent

Mutagen

any chemical agent that causes an increase in the rate of mutation above the normal spontaneous background rate

Single base changes

via deamination (turn C to U), problem because U is not base present in DNA

and alkylation (turn G to O6-methylguanine)

• alkylation commonly caused by chemicals, free radicals (cancer)

Structural Distortion

due to UV light - causes thymine thymine dimer, causes BULGE in helix

T-T dimer is major structural distortion

Intercalating agents

slot between H bonds and interrupt base pair stacking, cause structural distortion

DNA backbone damage

abasic sites and double strand breaks

abasic site- sugar/phosophate backbone lacking a nucleotide base

Responses to DNA damage

damage bypass, damage reversal, damage removal

Bypass via translesion synthesis

works around t-t dimer

error-prone DNA pol are recruited to bypass damage, they add anything opposite the dimer, causing an accumulation of point mutations or a frameshift mutation

Damage reversal NOT in mammals

photoreactivation - when t-t dimer is caused by UV light, photolyase binds to damage site and breaks bond when visible light is avaliable

Reversal of aklylation to O6-methylguanine

use methyltransferase to remove methyl from AA

methyltransferase is NOT an enzyme, it ends up changed, can only undergo one reaction (this is an energy demanding process)

DNA removal

involved multiple protein complexes, DNA travels from one complex to another

complexes have specific domains with specific functions

Base Excision Repair

repairs DNA when the base is damaged

Enzymes used in base excision repair

DNA Glycosylase recognizes and excises the damaged base

ex. hOOG1

Endonuclease removes nt near abasic site

DNA pol replaces messing nt

DNA ligase seals gap

Scanning DNA

function of DNA glycosylase to look for damage

occurs via simple diffusion, recognizes 8-oxoG while sliding over

Mismatch Repair

corrects mismatched bp at the end of replication

Hereditary non-polyposis colon cancer

inherits one inactive mismatch repair allele and the healthy/wild-type one is lost

more likely in cells that rapidily divide

Nucleotide excision repair (damage = T-T dimer)

XPC Scanning Complex looks for damage

converts to Interrogation Complex, opens DNA via twist-open mechanism

if it senses structural distortion, becomes Recognition Complex

Recognition complex and fixing damage

opens t-t dimer, inserts hairpin domain and flips out the damaged nt

helicase unwindes duplex and nucleases remove nt

gap is dna is filled by repair synthesis (clamp, loader, dna pol enzymes, primer)

dna ligase seals gap

Xeroderma Pigmentosum

defect in 😝 genes makes cell unable to complete nucleotide excision repair effectively

Repairing a double strand break in DNA

break caused by ionizing radicals or chemicals

fix via homologous recombination or non-homologous end joining

Homologous recombination

recognize damage and process ends

get genetic info from the undamaged homologous chromosome

strand inversion, branch migration

holidday junction resolution and ligation via RESOLVASE

Non-homologous end joining

error prone (indels can occur)

rejoin break via direct ligation of DNA ends

damage recognition + end processing (nuclease activity trims end)

then bridging and ligation

Hereditary breast cancer

due to mutations in BRCA1 and BRCA2 tumor supressor genes