Module 2: Molecular Basis of Inheritance and DNA Mutations Flashcards
What are the 3 functions of Genetic material?
Genotypic (replication)
Phenotypic (gene expression)
Evolutionary (mutation)
What are chromosomes made of?
DNA and proteins (sometimes RNA)
What is the difference between DNA and RNA
2nd C in DNA has H, 2nd C in RNA has OH - making RNA more reactive and degrades faster
What Nucleotides are Pyramidines?
Uracil, Thymine and Cytosine
What Nucleotides are Puriens?
Adenine and Guanine
What is the difference between A-T and C-G pairings
A-T have 2 H bonds, G-C have 3, making them stronger
What bonds are there in the DNA double helix?
- Covalent bonds in backbone
- H bonds between bases
- Hydrophobic bonds of bases in interior
What are the 2 flows of genetic information?
DNA –> DNA
DNA –> RNA –> Proteins
What did the Sia and Dawson experiment discover about genetic information?
Discovered that DNA carries genetic information by a series of experiments where they destroyed DNA, RNA and Proteins, and only when the DNA was destroyed was the genetic information not passed on.
What are the purposes of the 4 regions in a gene?
- regulation of transcription
- Regulation of translation
- Coding region - Usually starts with ATG
- Signal for termination of transcription - Usually TAA
How to the major and minor grooves of DNA double helix differ?
Major groove is wider and is also where proteins bind easier.
What are the 3 alternative forms of DNA?
A, B and Z
A and B are RIGHT HANDED
- B is more compact than A, fewer bases per turn and smaller diameter
Z is LEFT HANDED (Only seen in labs)
How does super-coiling of DNA occur?
Enzymes cleave one half of the DNA backbone - twist DNA then reseal to form a turn.
Most commonly forms L) handed turns - opposite to DNA helix.
What amino acids are most common in Histones and why?
Lysine and Arginine are most common because they have (+) charge, and therefore interact more with negatively charged DNA.
Define nucleosome
Bead structure of chromatin subunit - DNA wrapped twice (166 nucleotides long) around protein octamer (8 protein complex)
When do nucleosomes form?
During Interpahse
How does modifying proteins in the nucleosome octet affect then nucleosome?
More positively charged side chains –> more condensed DNA
What are the 3 levels of DNA condensation and when do they occur?
DNA (2nm) –> Interphase (11nm) –> Metaphase (30nm)
What are the 2 main models of 30nm fibre?
Solenoid (nucleosomes on same side of DNA linker - all pointing up) and Zig Zag (nucleosomes on alternating sides of DNA linkers - one up, one down etc.)
How does the structure of chomosomes form?
30nm fibre is packed in solenoid or zig zag formation then non-histone proteins act as scaffold to hold in x structure of chromosome
What are the 3 levels of DNA packaging in Eukaryotic cells
Interphase: 11nm fibre
Interphase and Metaphase: 11nm fibre folded and supercoiled into 30nm fibre
Metaphase: Scaffold proteins and 30nm fibre form Metaphase chromosomes
What is a centromere?
Constricted region of chromosome where spindle fibres attach. Primary consists of a satelite sequence alpha (171 base-pair-long sequence repeated over and over)
What is a telomere?
Sequence at the end of 3’ DNA that surpasses 5’ end of paired single strand DNA. Forms T LOOP to protect end of DNA and to prevent fusion of DNA.
Also facilitates the complete replication of the 5’ end of linear DNA molecules
What is the telomere sequence in vertebrates?
TTAGGG
What are the roles of TRF1 and TRF2 in the telomere loop?
TRF1 - separates 2 strands of DNA so overhang can loop back and connect.
TRF2 - reconnects DNA strand
What are the 3 steps of DNA replication?
Initiation
Elongation
Termination
What does it mean that DNA is semi-conservative?
Bot strands of parent DNA are conserved to form half of daughter DNA
Where does DNA start?
At the origin of Replication (site that has high A-T percentage)
Define replicon
Unit of replicated DNA controlled by one origin of replication. (Bacteria only have 1)
What is the role of DNA Polymerase III?
Adds nucelotides to leading and lagging strands of DNA. Primary Enzyme in DNA replication.
Adds DNA in 5’ to 3’ direction.
What is the role of DNA Polymerase I?
Removes RNA primers and replaces with required nucleotides. Corrects pairing inconsistencies. Can remove nucleotides in 5’ to 3’ AND 3’ to 5’ direction.
What is the process of DNA Replication?
1) Gyrase/Topoisomerase cuts DNA and Gyrase and Helicase unwind into 2 strands.
2) Single stranded binding proteins stabilise single strands - prevent binding to itself
3) Enzyme primase synthesises RNA primer
4) DNA polymersase III adds nucleotides to leading and lagging strands
5) RNA primer removed by DNA polymerase I, gap filled with nucleotides.
6) Ligase binds to OH, seals the backbone
What is needed for DNA synthesis
- Template DNA
- Primer
- Nucleotides
- Enzyme (DNA polymerase)
How does Eukaryotic DNA replication differ from Prokaryotic
- Shorter RNA primers and Okazaki fragments
- More origins of replication
- Nucleosomes must be unwound for replication
- Telomeres
When does DNA replicate in eukaryotic cells?
S phase
What are the important accessory proteins?
Dislodge then re-add histones
DNA Polymerase, Primase
PCNA (Proliverating cell nuclear antigen) - sliding clamp, moves forward on leading strand and helps repair.
Replication factor-c (RF-c) - loading of PCNA
Ribonuclease H1 and Ribonuclease FEN1 - removal of RNA primers
How does telomere replication affect Eukaryotic DNA replication?
Telomeres are at 3’ end of DNA that overhang, are a repeating series of nucleotides that gets shorter with each replication. Telomeres form a loop to protect the end from neucleases.
What is the role of telomerase?
To extend the telomere in germ line and cancer cells so they can replicate without being degraded
What are the primary transcripts of eukaryotic and prokaryotic DNA
Prokaryotic - mRNA
Eukaryotic - pre-mRNA (is then modified at both ends and introns removed for mRNA)
Where does transcription and translation occur in eukaryotic cells?
Nucleus: Primary transcription (pre-mRNA) - is modified at both ends and introns removed to form mRNA
Cytoplasm: mRNA is exported and translated by ribosomes
What is the role of RNA editing in eukaryotic cells?
Allows eukaryotic genes to form > 1 protein.
Why does protein synthesis take longer in eukaryotic cells than prokaryotic cells.
In prokaryotic transcription and translation can occur simultaneously, in eukaryotic it is 2 separate processes that occur in separate regions.
What is the difference between RNA synthesis and DNA synthesis?
- RNA precursers are ribonucleotide triphosphates (not deoxyribo-)
- Only 1 strand of DNA used as template
- RNA chain can be initiated de novo (without primer)
What is a transcription bubble?
A locally unwound segment of DNA in which transcription occurs
What is the process of transcription in prokaryotes?
1) RNA chain initiation - formation of transcription bubble
2) RNA chain elongation - adding of ribonucleotides to form mRNA chain
3) RNA chain termination - termination of transcription and release of nascent RNA molecule
What is a promotor in transcription?
A sequence of nucleotides tthat RNA polymerase recognises and attaches to to initiate transcription.
What are 2 important points in Prokaryotic Promotors?
- 10 - contains TATA (point where DNA starts unwinding)
- 35 - is recognised and bound by different transcription factors.
What are transcription factors?
Proteins that are different at different times that recognise different -35 sections
RNA polymerase binds to transcription factors - NOT DNA
What occurs during Initiation phase of RNA transcription?
1) Transcription factor recognises -35 section
2) RNA polymease binds to transcription factor
a. unwinds DNA
Why is there a higher rate of mutation in RNA?
RNA polymerase does not proof read - explains high mutation rate
What are the two types of termination signals in E. coli?
- Tho-dependent - require a protein factor (p)
- Tho-independent - not requiring (p) - Is more understood, a GC rich region that forms a hairpin that impedes
What are the additional features of the transcription process of Eukaryoties?
- 7-methylated guanidine cap added to 5’ of primary transcript
- Poly(A) tail added to the 3’ of the primary transcript
- Removal of intron sequences and RNA splicing
What is the role of enhancer sites?
Are prior to the -35 site and increase gene expression
Which RNA polymerase is most important to transcription?
RNA polymerase II - transcribes mRNA
What is the role of RNA polymerase III?
synthesis of tRNAs
What is the role of 7-methyl Guanosine
Contains a guanosine that is mehtylated at position 7 and is upside down
- Prevents degredation of 5’ end of RNA by RNases
- Is recognised by proteins that initiate translation of RNA –> Proteins
- Endonuclease site - proteins recognise and cut mRNA there
What is the role of the 3’ Poly(A) tail?
Added to 3’ end of mRNA by polyAmerase. Is up to 200bps long and protects the functional part of RNA from RNases
What are the 3 types of RNA involved in Translation?
mRNA
tRNA (transfer RNA)
Ribosomes (RNA and protein complex - 3-5 rRNA molecules)
What are the steps in translation?
1) tRNA charging
2) Initiation
3) Elongation
4) termination
What is the role of transfer RNA?
amino acids attach to tRNA by bonds between carboxyl group of acid and 3’ hydroxyl termini of tRNA.
tRNA recognise codons in mRNA so correct amino acid can bond to peptide.
Describe tRNA charging
1) ATP and amino acid react to generate aminocyl-AMP
2) Aminocyl-AMP is transferred to the tRNA 3’ end and AMP is released
What is the usual start codon, and what does it code for?
AUG - Methionine
What are the 3 stop codons?
UUA, UAG or UGA
Desciribe the structure of Ribosomes and the functions of its parts?
RIbosomes have large and small subunits of RNA and proteins
Small subunit binds at AUG
Large subunit binds to small subunit - polypeptide synthesised in large subunit
What are the 3 important sites on ribosomes
A site: amino acyl site binds to incoming aminoacyl-tRNA (carrying next amino acid to be added to polypeptide chain)
P site: peptide site binds the tRNA to which the growing polypeptide is attached. Is where peptide bond to tRNA is cleaved.
E site: Exit site - where uncharged tRNA leaves.
What is the Kozak sequence?
AUG - Eukaryotic initiation codon
What is the termination sequence for translation?
Release factor binds to stop codon
Breaks apart large and small subunits of ribosome
H2O is added to carboxyl terminus of peptide –> termination
What are the features of the genetic code?
- Composed of nucleotide triplets
- Non-overlapping
- Comma-free
- Degenerate (more than one codon per amino acid)
- Ordered
- Contains start and stop codons
- Nearly universal
Define Mutation
- change in genetic material
- The process by which change occurs
What are the features of mutation?
1) Somatic or Germinal
2) Spontaneous or Induced
3) Usually random/non-adaptive
4) Adaptive or stationary phases (based on environmental stress)
5) REversible
Define mutatnt
Organism that exhibits a novel phenotype
Define mutation
heritable changes in genetic material that provide the raw mutation for evolution
What is the difference between germline and somatic mutations/
Germinal - occurs in germ-line cells. Usually mutation is not seen in first generation. Heritable.
Somatic - mutant phenotype only occurs in descendant of that cell, is not heritable.
Define spontaneous mutation
Occurs without a known cause due to inherent metabolic errors or unknown agents in the environment
Define Induce mutation
Result from exposure to mutagens, physical or chemical agents that cause change in DNA
Describe the adaptive and stationary phases of bacteria.
During low environmental stress population grows, low rate of mutation.
Higher environmental stress - population plateaus, higher rate of mutation
What are the two processes of reversing mutation?
1) back mutation - second mutation at the same location. All offspring will be wild tyle
2) supressor mutation - second mutation at different location. Some offspring will still have mutation.
Define Null allel
No protein is produced –> phenotype normally present is absent
Define isoallele
no effect on phenotype, or small effect only identifiable by a special technique
What is a tautomeric shift?
A point mutation occuring during DNA replication
- H atom moved form one position on Purine or prymidine ring to another position
- Allows C-A (rare imino form) or G-T (rair enol forms) to pair
What are the types of base substitution?
Transition: prymidine with prymidine, purine with purine
Trnasversion (uncommon): prymidine with purine etc.
What are frameshift mutation
Only occur in coding regions, due to addition or subtraction mutations
Affect protein structure and function
Define induced mutation
Following exposure to physical or chemical mutagens
What are the types of chemical mutagens
- Mutagenic ONLY to replicating DNA (Base analogs and acridine dyes)
- Mutagenic to replicating and non-replicating DNA
What are mutagenic base analogs?
molecules with similar structure to normal bases, however their differences increase the likelihood of mispairing/mutations
E.g. 5-bromouracil (thymine analog) - unstable pairs G, introduces point mutations
What mutation does nucleic acid induce?
Removal of amino group from bases.
What mutation do alkylating agents induce?
Donate akyl to other molecules - induce mutations/
How doe UV light induce mutation?
Excites molecules - forms prymidine hydrates and prymidine dimers (T- dimers)
Expansion of Trinucleotide repeats mutation
Trinucleotide repeats (3 of same nucleotide pairings) can increase in copy number when loops form that DNA polymerase goes through > 1 time so repeat gets longer with each DNA replication - Causes diseases characterised by 'anticipation' where onset is earlier or severity increased with successive generations
Repeats induce a transposoin
Define Transposoin
DNA segment capable of replicating and inserting copies of DNA into random sites in the same or a different chromosome
What are the methods of DNA repair?
- Light dependant repair
- Excision repair
- Post-replication (including mismatch) repair
- Error-prone repair system (SOS response)
What does Light-dependant repair do?
Photolyase cleaves thymine dimers (is activated by blue light)
What is the process of excision repair?
Involves at least 3 steps
1) DNA repair endonuclease or endonuclease containing complex recognises, binds to and excises damaged base or bases
2) DNA polymerase fills gaps
3) DNA ligase seals the gap
What are the types of Excision repair?
- Base excision repair: removes abnormal or chemically modified bases
- Nucleotide excision repair: removes larger defects, e.g. thymine dimers
What initiates base excision repair?
DNA glycolases
What is the process of base excision repair?
1) modified base
2) Specific glycolase binds to specific altered base
3) bond between abnormal base and 2-deoxyribose cleaved - creates apurinic or apyramidinic site with missing base
4) AP site recognised by AP nucleases, that with phosphodiesterases excise the sugar phosphate groups at this site
5) DNA polymerase replases missing nucleotides
6) DNA ligase seals the nick
What is the role of Nucleotide excision repair?
Removes thymine dimers
What mechanism allos Post-replication mismatch repair to identify new vs parent strands of DNA?
shortly after synthesis DNA is methylated. Prior to this 4 genes (mutS, mutH, mutU and mutL) act together to remove errors in newly synthesised DNA
Error cleaved then gap filled by polymerase
Post replication repair of T dimers
Error identified and genes bond to it (e.g. gap in new DNA from T dimer where DNA polymerase unable to synthesise new strand)
Gap filled with original sister strand to error DNA
Gap filled in original sister strand by polymerase using new strand as template, ligase seals gap
What is the SOS response?
Occurs when DNA is heavily damaged
DNA polymerase V replicates DNA in damaged regions - is error prone/not accurate
Gene for SOS response is usually inactive - when DNA is damaged RecA bonds to single stranded regions of damaged DNA - activates response.
What is the role of the DNA recombination mechanism?
To induce genetic variation and repair mutations
Occurs during meiosis 1 and prophase 1
What is the process of DNA recombination?
1) Homologous chromosomes pair
2) Strands separate and swap over (2 chromosomes now connected)
3) RecA drives the process
4) DNA forms X structure - resolution occurs when DNA is cut and separate
What are the products of DNA recombination?
- Recombinant DNA - Each side of where strands were connected is from different chromosome, DNA swapped over due to how X structure was cut.
- Non-recombinant DNA - Chromosome is mostly the same, small section of DNA that belonged to other chromosome that matched the original nucleotide sequence from crossing over.
