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DNA REPLICATION AND REPAIR (DNA replication process (key step is…
DNA REPLICATION AND REPAIR
DNA replication
two anti-parallel strands of DNA are held together by hydrogen bonding
models considered to explain DNA replication
conservative
dispersive
semiconservative
DNA replication is semiconsevative
the Meseson-Stahl experiements
used the density of N to show DNA is semi conservative - original strand contains N15 (heavy) and newly synthesised strand contains N14 (light). Density shown using centrifuge tube.
the Taylor-Woods-Hughes experiment
as chromosomes enter mitosis, both chromatids of the chromosomes are radioactively labelled. After a second round of replication only one chromatid of each chromosome is radioactively labelled
experiment using mitosis - A) One round of chromosome replication in the presence of BrdU results in half-labelled DNA duplexes. (B) Two rounds result in one fully labelled duplex and one half-labelled duplex. (C) Full labelling quenches (dulls) staining, so that the half-labelled chromatid is dark but the fully labelled chromatid remains dull.
requirements for DNA replication
primer
mononucleotide phosphates
template
enzyme
DNA replication process
key step is elongation
inserts it across the template nucleotide; if it doesn’t match rejects it and chooses another one
hydrogen bonds are formed across the matching bases; the α phosphate group ‘attacks’ the 3’ OH group on the primer
the polymerase enzyme plucks a deoxy-nucleotide-triphosphate from the pool
the phospho-diester bond is formed between the 5’ of new nucleotide and the 3’ of the primer; the pyrophosphate is released
the DNA strands have to be separated
separation is the job of DNA helicase
unwinding generates torsional stress, which is removed by DNA topoisomerase 1 (in eukaryotes), or gyrase (in prokaryotes)
the two strands of DNA are in antiparallel orientation
one strand is synthesised continuously, leads the synthesis.
synthesis of the other strand lags behind, because every time new initiation has to take place
DNA synthesis is bi-directional
during 0-form replication, both parental DNA strands remain intact. Bacteria replicate their circular chromosomes via this method, mostly in a bidirectional way
forks moving in opposite directions form a bubble
at the fork moving to the right the top is the leading strand, the bottom is the lagging strand
at the fork moving to the left it is the other way round: the top is the lagging the bottom is the leading strand
DNA polymerases can’t initiate synthesis
template-dependence is how they COPY the template
primer-dependence needs a work-around: primase
DNA polymerase enzymes are primer and template dependent.
primase is a special RNA polymerase. RNA polymerases are NOT primer dependent
the RNA primer has to be removed: Okazaki fragment maturation
a. completion of Okazaki fragment synthesis leaves a nick between the Okazaki fragment and the preceding RNA primer on the lagging strand
b. DNA polymerase 1 extends the Okazaki fragment while its 5'→ 3' exonuclease activity removes the RNA primer. This process, called nick translation, results in movement of the nick along the lagging strand
c. DNA polymerase 1 dissociates after extending the Okazaki fragment 10 -12 nucleotides. DNA ligase binds to the nick.
d. DNA ligase catalyses formation of a phosphodiesterase linkage, which seals the nick, creating a continuous lagging strand. The enzyme then dissociates from the DNA.
incorporation must be faithful
an unpaired base activates the 3' to 5' exonuclease activity of DNA polymerase III
Okazaki fragments: short, newly synthesized DNA fragments that are formed on the lagging template strand during DNA replication
DNA replication
b. as helicase unwinds the DNA template, primase synthesises an RNA primer. The lagging strand polymerase completes an Okazaki fragment.
c. when the lagging strand polymerase encounters the preceding Okazaki fragments, it releases the lagging strand.
a. the lagging strand template loops back through the replisome so that the leading and lagging strands are synthesized in the same direction. SSB binds to single stranded DNA.
d. the lagging strand polymerase binds to a newly synthesised primer and begins synthesising another Okazaki fragment
some of the key proteins in DNA replication
DNA ligase: joins Okazaki fragments on lagging strand
Helicase: unwinds DNA at the replication fork
RNA primer: initiates new strand synthesis
SSB proteins bind and stabilise single stranded DNA at replication fork
gyrase: cleaves and swivels duplex to release mechanical stress of unwinding
primosome: protein complex responsible for creating RNA primers on single stranded DNA during DNA replication
DNA polymerases: enzymes that synthesize DNA molecules from deoxyribonucleotides, the building blocks of DNA
DNA damage
caused by a variety of sources
ionising radiation
chemical exposure
UV light exposure
replication errors
cellular metabolism
different outcomes
repair
programed cell death (apoptosis)
transcriptional programme activation
cell cycle checkpoint activation
direct damage to bases
deamination
spontaneous loss of the amino group of cytosine to yield uracil
spontaneous loss of the amino group of 5-methylcytosine to yield thymine
deamination of adenine results in hypoxanthine. Hypoxanthine base pairs with C
akylation
physiological methylation of cytosine at the number-5 position in the base
mutagenesis of guanine by ethyl methanesulfonate (EMS
oxidative damage
oxidation of guanine results in 8-oxoguanine. 8-oxoguanosine base pairs with A
main source: oxidative metabolism inside the cell
UV light exposure damage
causes two thymine's to move closer together forming a thymine dimer which causes distortion of the DNA helix
most common source of UV damage is overindulging under the sun
cancerous moles
ionising radiation ,
causes mostly single and double strand breaks
most common source of exposure in USA is radon gas
induction of mutations by radiation is linearly related to exposure dose
replication errors
the error rate can be once in every 10’000-1’000’000 incorporated nucleotides
this is improved enormously to 1/100’000’000 – 1/10’000’000’000 by the proofreading activity of the polymerase
the exonuclease subunit takes over and removes the misincorporated nucleotide
nevertheless, some mistakes can still be left behind
DNA's 'spelling mistakes'
mutations in NER genes are associated with three groups of diseases
Cockayne syndrome (CSA[ERCC8], CSB[ERCC6])
short stature
premature ageing
microcephaly, impaired CNS development
failure to gain weight and grow at expected rate
extreme photosensitivity
Trychothiodystrophy (ERCC2, ERCC3, GTF2H5)
brittle, sparse hair (in mild forms only this)
delayed development
significant intellectual disability
recurrent infections
about half of patients have the photosensitive form
incidence ~1:1’000’000; 100 reported cases
Xeroderma pigmentosum (XPA, B, C, D, E, F, G, ERCC2, ERCC3, POLH)
extreme photosensitivity
susceptibility to develop skin cancers (without sun protection from the age of 10!)
DNA repair
types of repair
nucleotide excision repair
mismatch repair
base excision repair
double strand break repair
direct reversal
homologous recombination
non-homologous end joining
process
detection eg by specific glycosylases
removal eg by glycosylases
patching up eg by DNA polymerase
sealing eg by ligase