high fidelity (but need mutations)

highly processive

relatively fast

both strands get a new stand

this is the correct method

-demonstradted semiconservative

-applied CsCl gradient ultracenterfugation of DNA labeled with Heavy Nitrogen (15N)

-cells were moved to growth medium containing normal Nitrogen (14N)

-DNA was isolated at different times

-pictures taken during centerfugation

-found old strand of heavy DNA with new strand of light DNA

both strands serve as templates in the same compass direction 

one would do 3'-->5' and hte other would do 5'-->3'

-proposed that DNA polymerase can make one strand of DNA continously 5'-->3' (leading strand)

-but the other strand would be discontinous in 5'-->3' (lagging strand)

-if short pieces of DNA are synthesized on the lagging strand, we should be able to catch them with radiolabeling

-if the enzyme DNA ligase is eliminated from the replication process, then the short pieces of DNA made ought to be detectable even at longer labeling periods

-used T4 phage mutant that made Ligase

-control - found short pieces for short periods of time before they were ligased together

-using the T4 mutant - tons of short pieces as time incresaed 

required for DNA synthesis to begin

laid down on the template strands to provide a double-stranded region for DNA polymerase to bind

bidirectional

simultaneously

2 replication forks

oriC

contains 4 9-mers having a consensus sequence of TTATCCACA

-discovered the theta mode

-labeled replication E. coli DNA with radioactive DNA precursor and observed with autoradiography 

-did not display bidirectional replication

-radiolabeled replicating DNA for a short time with low levels of radioactive precursors, followed by high levels of radioactive precursor

-the nucleotides began to be incorporated in the new strands of DNA created

-not the theta mode

-example of unidrectional

-occurs in phage φX174 and i'm guessing  λ phage

-one strand of dsDNA is nicked and 3' end is extended

-intact strand is used as template

-a single stranded circle of DNA is released as single stranded circle of DNA

-normally exists as ssDNA but has to be double stranded for replicative form

-strand nicked and 3' end laid out

-its this nicked strand that serves as template for discontinous lagging strand sythesis 

-an ATP-dependent enzyme that separates the DNA strands in advance of the replication fork

-the dnaB gene product in E.coli

bind to ssDNA and prevent it from reforming dsDNA

-product of ssb gene of E.coli

-the bonding is cooperative - raises affinity by 1,000 fold for next molecule

-stimulate replication

as the DNA unwinds, it has to wind somewhere upstream, creates strain

-topoisomerase releaves this strain by introducing temporary single- (Type I) or double-stranded (Type II) breaks in the DNA

from E. coli

type II topoisomerase

Polymerase I - DNA repair, primer excision

Polymerase II - SOS repair

Polymerase III - required for DNA replication in E.coli

Holoenzyme polymerase III subunits

aka: pol III holoenzyme

composed of multiple subunits

o   The α-subunit has the DNA polymerase activity

o   The ε-subunit has the 3’ à 5’ exonuclease proofreading activity

o   The θ-subunit has the ???? activity. The function is still unknown

o   DNA polymerase α (priming)

o   DNA polymerase  δ (elongation)

o   DNA polymerase β (repair)

o   DNA polymerase ε (repair)

o   DNA polymerase γ (mitochondrial)

inition

elongation

termination

-binds at the oriC

-facilitates the binding of dnaB

-stimulates melting of the 3 13-mer repeats at one end of the oriC to make an opening 

-required for protein synthesis

-dnaC binds to dnaB

-stilumates binding of the primase

-also serves as the helicase that moves 5'-->3' on the lagging strand in the direction of the replication fork

-RNA polymerase which synthesizes a short piece of RNA that creates an R loop

-helix unwinding (HU) protein which induces bending

-product of the dnaG

-its the RNA primer-synthesizing enzyme 

primosome 

-this is responsible for laying down multiple primers for Okazaki fragments on the lagging strand 

-multiple sites of replication for each chromosome

-and that's about all we know

due to the sliding clamp

-its the β-subunit of the holoenzyme

-literally holds the entire pol III assembly on the template for long periods

the γ-complex

-sliding clamp cannot touch the DNA by itself

-serves as the clamp loader and is ATP-dependent

processivity factor PCNA

PCNA = proliferating cell nuclear antigen

-forms a trimer (3 subunits) that can encircle the DNA as the bacterial clamp does

-contains two core polymerases - one for each strand

-as it finishes one Okazaki fragment, it runs into a nick that is positioned in front of the primer on the next fragment 

- this nick is a cue for the complex to dissociate from the and move to the primer on the next fragment 

terminus

-where the replication forks begin to get near each other

-contains 22bp sites that bind specific proteins called TUS proteins

-replication forks stop moving when they get to this region

-leaves the daughter duples entangled 

-process of untangling the interlocking DNA rings

-preformed by topoisomerase IV

-there are gaps left when RNA primers are removed

-this is problematic because when the primer is removed DNA cannot be extended in the 3'-->5' direction

- and there's no 3' upstream

-so DNA should be getting shorter...

worked with Tetrahymena

discovered telomers

-repeats of GC-rich sequences at the ends of eukaryotic chromosomes

-telomerase adds these strands, not DNA polymerase 

-exact sequence of the telomere is species specific and is also determined by the small bit of RNA inside the telomerase

-carries a small RNA as part of its structure and the RNA serves as the template for telomere synthesis

-it is a ribonucleoprotein

terminaton - which is which

TTGGGG

TTAGGG

TTGGGG - Tetrahymena

TTAGGG - vertebrates

-Hayflick (1960s) showed that normal animal cell lines are not immortal

-grow in cultures of about 50 generations, then enter senescence

-cancer cells do not have this limit (contain telomerase as do egg and sperm production cells)

-not the same thing

-damage is a chemical alteration

-mutation is a change in a base pair

-unrepaired damage can lead to mutations

-alkylation of bases

-UV-induced Cross Linking 

-Ionizing radiation

-phosphate groups

-N7 of guanine

-N3 of adenine

-O6 of quanine but is rare

-creates 3-methyl adenine (3mA) that cannot base pair properly with any other base

-the 3mA is a noncoding base

-DNA polymerase stalls at it - doesn't compute 

-polymerase can go on and skip, but can result in erroneous replication which leads to mutations

-seldom but very mutagenic when it does

-the product can base pair with T instead of C

-occurs from the introduction of ethylmethane sulfonate

-pyrimidine dimers by cross linking adjecent pryimidines on the same DNA strand 

-and is pretty common

-and they block DNA replication

-pretty much 2 Ts melt together so they look like one... and only 1 A is given to them

the 2 bonds that link the thymines form a four-memebered cyclobutane ring

and that's all i have 

-high energy x-rays and gamma radiation

-also called high energy radiation

-primarily caused by free radicals

-form when water is ionized by the incident radiation

-have an unpaired electron and are extremely reactive

-especially the ones that contain O

-breaks dsDNA

-which this does

-difficult to repair 

-directly repairing the damage

-removing the damage and replacing it with new DNA

-light repair

-not in placental mammals but in all others...

-carried out by DNA Photolyase

-recognizes and binds to the pryimidine dimers

-is activated when it absorvs light energy in the UV-A/blue (>300nm) part of the spectrum

-breaks the dimer bonds to reform monomer

-for the alkylation damage of O6 guanine

- also called O6-methylguanine methyltransferase

-the sulfur atom of a cysteine in the enzyme accepts the methyl or ethyl alkyl group

-this irreversibly inactivates the enzyme

-transferase is induced by DNA alkylation

base excission repair (BER)

nucleotide excission repair

DNA Glycosylase

AP Endonuclease

DNA Phosphodiesterase

DNA Polymerase I

DNA Ligase

Apyrimic or Apurinic

means without the bit it is missing lol 

Global Genome NER

Transcription-Coupled NER

-confined or restricted to transcribed parts of the genome

-only occurs in parts of the genome being actively transcribed

-RNA polymerase will stall at the lesion leaving a region of melted DNA

-will work on any DNA within the genome

- repair to any lesion damage throughout the genome can occur

-Xerroderma Pigentosum

-thousands of times more likely to get skin cancer

-helix distorting damage such as dimmer formation goes unrepaired due to the defective NER

-recognizes the distortion in the helix and binds to it

-induces localized DNA melting around damage

-RPA binds to the undamaged DNA across from it

-TFIIH, a helicase, causes additional melting

-RPA helps position 2 endonucleases (ERCCI-XPF complex and XPG) on either side

-an oglionucleotide containing the damage is excised

-DNA polymerase ε or δ fills the gap

-ligase seals the nick