DNA Biochemistry Flashcards
Lecture Outcomes
- List hypotheses on the origin of life on earth.
- Know the main events which led to the discovery of DNA.
- Be able to describe the main features of DNA.
- Understand how the genetic code works.
- Know what is meant by “The Central Dogma”.
- Know the terminology for bases, nucleotides, nucleosides, deoxy, ribo.
- Be able to define the terms antiparallel, complementary base pairing, coding strand, codon, right-handed helix, major groove.
Four hypotheses on origin of life on earth
- Organic chemical synthesis in a reducing atmosphere
- Carriage by meteorites
- Organic chemical
synthesis
deep ocean vents - RNA world
- Organic chemical synthesis in reducing
atmosphere
- Was thought that early earth had
a reducing atmosphere, rich in hydrogen and methane. - Miller, S.L. (1953) subjected methane, ammonia & hydrogen gas
mixture to electrical discharges presence of water (famous Miller-Urey experiment).
Prebiotic soup resulted (amino acids and nucleotides).
- No data on how soup forms organic networks encompassed by a membrane.
- But was primitive atmosphere reducing? Current consensus is that it was not.
- Carriage by meteorites/comets
- “Panspermia” - attractive theory due to sudden appearance of life on earth and its amazing uniformity (but no data).
> Organic compounds common in space.
Amino ace sanine fundin comet 2088. Than, based on studies simulating its atmosphere (2013, NASA).
- Mars rocks blasted into space by meteor impacts, carrying microbes?
- Only moves the question backwards - how did life originate
e sewnere.
- Synthesis on metal sulphides in deep
sea vents
- Vents are sites of abundant biological activity, much of it independent of solar energy.
- Energy source, chemical source leads to another prebiotic soup.
- Prebiotic soup self-organizes into life-supporting networks on metal sulphide surfaces.
- Networks incorporate into membranes (no data).
Discovering the DNA structure
Watson and Crick 1953
The Cavendish Laboratory, Cambridge UK.
- RNA world
- Was the first self-replicating entity simpler than a cell?
- Short RNA molecules were discovered that can store information and catalyse chemical reactions (ribozymes)
- RNA molecules have been synthesised that are capable of self-replication
- How did lipid membrane form around RNA?
Behind the Discovery
- James Watson sees X ray diffraction image of DNA shown by Maurice Wilkins (Kings College London) at conference in Naples.
- Frances Crick works on helical diffraction in proteins in the same laboratory.
- November 1951 better X ray data from Rosalind Franklin (Kings College).
- Watson and Crick produce a three-stranded DNA model.
- Franklin points out this model’s inconsistencies with her data.
Moving back & Moving on
- DNA is the genetic material
- DNA is a base-paired, anti-parallel, right-handed double helix
- The code is cracked (triplets of A, T, G and C code for individual amino acids, the building blocks of proteins
- Gene to protein relationships established
- Control of gene expression partly elucidated
- Large scale sequencing of genomes now common
How does the genetic code work?
Taken three at a time, combinations
64 are possible, which is enough to characterise the 22 amino acids plus ‘stop’.
How does the genetic code work?
- Backwards explanation with an example
- Highly active neuropeptide present in human brains: met-enkephalin
- The amino acid sequence is
(N) Met Tyr Gly Gly Phe Met (C) - The DNA code is
(5’) ATG TAT GGT GGT TTT ATG (3’)
How does the DNA code look in context?
The Central Dogma/Overview photo
Nucleotides: The building blocks of
DNA/RNA photo
Terminology Continued
- If sugar is deoxyribose, prefix names with deoxy
-ex - Deoxyadenosine monophosphate (dAMP)
- Deoxyadenosine triphosphate (dATP)
- If sugar is ribose, prefix names with ribo - Riboadenosine triphosphate (rATP)
- DNA (deoxyribonucleic acid) contains deoxyribose
- RNA (ribonucleic acid) contains ribose
deoxyribose sugar photo
Terminology for nucleotides and
nucleosides photo
How The Chain Is Linked photo
Important features to remember:
Important features to remember:
- Strands are opposite directions (i.e. ANTIPARALLEL)
- Strands are COMPLEMENTARY. Sequence of one strand defines the seauence of the other strand from base pairing rules (A=T & G=C)
- Information encoded by order of bases 5’ to 3’ One CODING strand & other is NON-CODING
- THREE bases = ONE codon (i.e. codes for 1 amino acid) e.g. ACG encodes for threonine, TTC encodes for phenylalanine
Important features to remember:
dna photo
DNA Helix is Right-handed photo
What’s happening with DNA now?
- Genes have been/are being patented
> Update: in 2013 the US Supreme Court has ruled that human
genes cannot be patented
- “Junk DNA” has been patented
> (87% of the human genome)|
- Transgenics and gene KO/KI developed
- Knock-out mutants: loss/inactivation of gene
> Knock-in mutants: addition of gene
- Genetic screening moves into medicine
- Viruses and living cells created from synthetic DNA constructs
- “Bioinformatics” is born
DNA interaction with Proteins
Proteins can interact with bases in “major groove”
Proteins can recognise specific base sequences
Is there still research on DNA?
- “Junk DNA” is not junk
- DNA can change to other forms (Z and G) in vivo and such changes alter gene expression
- Z DNA has a left-handed helix
- Chromosomal position and movement within the nucleus is preserved across species and affects gene expression
- Confocal microscopy of living cells reveals DNA in real-time as a “demonic dancer”
Lecture Outcomes:
- Define the terms describing DNA replication: semiconservative, origin, bidirectional, replication fork, Okazaki fragment.
- Understand the mechanism of leading and lagging strand replication and role of the RNA primer.
- Understand the functions of the proteins at the DNA replication fork.
- List major DNA polymerases of prokaryotes and eukaryotes and their functions.
general features applying to all chromosome replication
- Complementary base-pairing enables SEMICONSERVATIVE DNA
replication - DNA synthesis initiates at ORIGINS
- Synthesis usually moves BIDIRECTIONALLY away from an origin via two REPLICATION FORKS, thus producing a REPLICATION
BUBBLE - Synthesis of new DNA is always 5’→ 3’
- Synthesis of new DNA always requires a PRIMER
Complementary base-pairing enables accurate DNA replication
- Each strand of a dsDNA molecule serves as a template for synthesis of a new complementary strand
- A binds only with T
- G binds only with C
DNA replication is semiconservative
- Each strand of a dsDNA molecule serves as a template for synthesis of a new complementary strand
- Each daughter molecule has a parental strand plus a new strand
- Accuracy and speed - 1000 nucleotides per second without error
DNA synthesis initiates from origins (ori)
- dsDNA pried apart at replication origin by helicase, at position identified by particular DNA sequence = ori
- Group of proteins meet to operate as a protein machine moving along
replication fork - DNA polymerase adds nucleotides to 3’ end of new strand
- DNA polymerase has proofreading property to reduce error rate
bidirectional synthesis from origins
- Circular (short) chromosomes of prokaryotes e.g. E. coli have a single origin of replication
Parental strands orange, new strands red → direction fork is moving
bidirectional synthesis from origins photo
Bidirectional synthesis from origins photo
Structure of a replication fork
- Both daughter strands polymerized in 5’ to 3’ direction
- Leading strand is synthesized continuously
- Lagging strand is synthesized discontinuously, made as series of short Okazaki fragments
Structure of a replication fork
- New strands are synthesized in the 5’ to 3’ direction.
- The lagging strand of DNA must be made initially as a series of short DNA strands called Okazaki fragments, these are later joined together.
- DNA strand that is synthesized discontinuously is called the lagging strand.
- The other strand is synthesized
continuously and is called the leading strand.
DNA synthesis is catalysed by DNA
polymerase
- DNA polymerase adds each deoxyribonucleotide to the 3’ end of a primer strand attached to the template strand.
DNA synthesis is catalysed by DNA
polymerase photo
IDK why but its always called deoxyribonucleoside triphosphate and never deoxyribonucleotide triphosphate
Primers for DNA synthesis
- DNA primase is an enzyme that synthesizes a short strand of RNA on a DNA template
- During lagging strand synthesis, each Okazaki fragment is primed by an RNA primer, which is synthesized in a template dependent manner by DNA primase
- DNA ligase joins fragments by their sugar-phosphate backbones
The proteins at a replication fork cooperate to form a replication machine
The proteins at a replication fork cooperate to form a replication machine
- Single-strand DNA-binding proteins stabilise ssDNA and aid the helicase
- Helicase pries apart (unwinds) the double helix to form ssDNA for replication
- Sliding clamp holds DNA polymerase firmly on the DNA during DNA replication
- Clamp loader assembles the clamp on the DNA using ATP energy
Current view of the arrangement of replication fork
Lagging strand DNA is folded to bring its DNA polymerase into a complex with leading strand DNA polymerase.
Current view of the arrangement of replication fork
DNA polymerases of Escherichia coli photo
DNA polymerases in mammals photo
Lecture Outcomes
- Explain mutation of DNA
- Describe the processes that result in the mutation of DNA
- Describe the consequences of depurination, deanimation, thymine dimer formation and double stranded breaks on DNA replication
- Understand how transposable DNA elements and infectious agents introduce mutations into DNA
- Explain the two mechanisms for DNA repair: MisMatch repair system and homologous recombination
- Definition of Mutation
Any permanent and heritable change in the DNA sequence
of an organism
- Consequences
Damaged DNA will cause problems with DNA replication, lethal
- Repair restores DNA replication
Restoration of correct nucleotide sequence
Repair can result in incorrect nitrogenous base being incorporated
- To overcome these problems, all living cells have mechanisms for DNA repair
- How do changes in DNA sequence occur?
‒ Replication errors (Very Rare)
DNA replication is referred to as
“HIGH FIDELITY”
Incorrect copying by DNA polymerases results in only 1
error in 1,000,000,000 bases (1:109 or one in a billion)
- This high fidelity is caused by:
‒ Base paired structure of DNA
‒ The primer requirements of all DNA polymerases
‒ The “proof-reading” of DNA polymerases
fidelity pho5o
Mutation of DNA: Environmental factors photo
Mutation of DNA: Nucleotide Instability photo
Mutation of DNA: Nucleotide Instability
remember the consequences
Outcome: DNA polymerase randomly assigns nucleotides to match damaged nucleotide, therefore, a change in the nucleotide sequence becomes fixed and inherited.
Mutation of DNA: Nucleotide Instability photo
Mutation of DNA: Mutagenic Chemical
Alkylation
Electrophiles add alkyl groups to nitrogenous bases, stalls replication
eg. carcinogens, methylmethane sulphonate (MEMS)
Mutation of DNA: Mutagenic Chemical
Intercalation
Compound inserts into the double stranded helix leading to distortion
Does not change the bases
eg. Ethidium bromide
Mutation of DNA: UV Light photo
Mutation of DNA: Other forms of Radiation
- Gamma and X-rays
‒ Attack DNA bonds by:
- directly producing free electrons
which attack DNA backbone - OR indirectly by generating hydroxide free radicals
‒ Both result in single and double stranded breaks
Mutation of DNA: Other forms of Radiation photo
Mutation of DNA: Infectious Agents
- Mobile DNA, has the ability to insert or recombine into a target DNA molecule e.g:
Infectious agents eg viruses, bacteriophages
Transposons
- Recombination, is the breaking and rejoining of DNA molecules to form new combinations
Non-homologous recombination means no similarity between DNA molecules is required
o Site-specific (or targeted) recombination catalysed by enzymes called integrases and transposases
Homologous recombination means both the donor and acceptor DNA molecules have extensive similarity in DNA sequences
Mutation of DNA: Infectious Agents
- Retroviruses such as HIV (cause of AIDS) and their equivalents in bacteria (bacteriophages) can integrate into host DNA
- Parasites that utilize the host cell replication machinery
- Lytic (enter and lyse host) and lysogenic (enter and integrate into host chromosome) life cycles
- Insertion of foreign DNA physically disrupts a coding region (eg. Gene)
HIV prefers to integrate into transcriptionally active genes
- Not a common feature of all viruses
Mutation of DNA: Transposons
- Transposons
Linear DNA molecule
move within and between chromosomes
insert into many different DNA sequences
- Consequences
Insertion into a gene will physically disrupt it
Excision of transposon can result in small duplication of DNA - again disrupting a gene
Common in bacterial DNA rearrangement
Mutation of DNA: Transposons photo
DNA repair: Basic Mismatch repair
- DNA replication without mismatch repair - 1 mistake per 107 nucleotides copied
- DNA replication with mismatch repair - 1 mistake per 10° nucleotides copied
- “Mismatch” refers to mis-paired nucleotides
- Repair - repair proteins recognise and excise strand of DNA containing the mismatch
Mutation of DNA: Transposons photo 2
Non-homologous recombination:
Transposition
Non-homologous recombination:
photo
Non-homologous recombination:
Non-homologous recombination between sites on bacterial DNA and phage DNA
- Phage encoded Int (integrase) protein promotes recombination between the attachment sites (att), attP and attB
DNA repair: Basic Mismatch repair steps
- Four steps:
- The repair proteins patrol the DNA and bind to the mismatched sequence
- The mis-matched region is excised by nucleases, thus creating ssDNA patch
> Synthesis of the second strand by repair DNA polymerase using the free
3’OH group as a primer
- Ligation of the DNA backbone by DNA ligase
Repair mechanisms result in the restoration of the original sequence, two examples:
- Mis-Match repair system repairs mutations in the newly synthesised DNA strand to restore it to the original sequence of the template strand
- Homologous recombination repairs double-stranded breaks in the phosphodiester backbone of the DNA
- this may result in restoration of the original sequence but can also result in rearrangements of local regions of the DNA sequence
DNA repair: Basic Mismatch repair steps
Homologous recombination
- Double stranded break introduced into chromosome A
- Exonuclease removes nucleotides from the 5’ to 3’ direction
- The single stranded 3’ overhang can migrate into the recipient chromosome B where the sequences are homologous
Repair of DNA by Homologous
Recombination
- Homologous recombination
- Regions of very similar sequences align
- Double strands are broken then a cross over occurs
- DNA repair, generate new combinations of DNA
Homologous recombination photo
Homologous recombination continued
- DNA polymerase synthesises new complementary strands
- Crossed strands also known as Holliday junction
- Rotation of crossed strands to allow section of one strand to be joined to section of another strand (exchange of DNA)
- Nucleotide sequence at site of exchange is unaltered (no additions or subtractions)
Homologous recombination continued photo
