PL section - DNA replication Flashcards
Which gene is one of the most well stored in DNA?
ribosomal gene
(importance of protein synthesis)
How is DNA information stored from kept from cell to cell?
by DNA replication
What are 3 necessary things for DNA replication?
- Primer sequence (DNA pol can’t start new strand, can only elongate)
- Pool of unincorporated nucleotides
- Proteins that collaborate to catalyse
What reaction starts the addition of a new nucleotide to the DNA chain?
- Matching in the base pairs between template and incoming dNTP
- Polymerization (3’ OH attacks alpha-phosphate of incoming dNTP)
What is the replication fork?
Area where nucleotides are added, where DNA parent duplex separate
Replication fork moves along the double helix as replication occurs
What is the role of DNA helicase in the DNA replication?
Allows parent DNA duplex strands to separate
What is a Primer?
Primer = Short RNA molecule complementary to single-stranded region of DNA parent
(Because DNA pol cannot initiate synthesis of a new strand)
What is Primase?
It an enzyme, a specialized RNA polymerase which forms a NEW complementary RNA molecule which will be elongated for DNA replication
What are the Leading and Lagging strands?
Leading strand = side where new strand in synthesized continuously from 5’ to 3’ (adding on 3’ end, which is at the replication fork)
Lagging strand: replication fork is at 5’ end so not continuous synthesis
Okazaki fragments are synthesized with multiple short RNA primers (starting at replication fork and going to next RNA primer)
What are Okazaki segments?
Short discontinuous fragments consisting of RNA and DNA
What is the role of DNA ligase?
It joins 2 Okazaki segments (after RNA primer was replaced by DNA)
What is the replisome?
The molecular machine involved in DNA replication
On what organisms were discovered much of what we know about DNA replication?
Prokaryotes (ex: E. coli)
Viruses (SV40) bc viruses attack host and uses the host’s mechanisms so easier to study them
What is the large T-antigen ?
It is a protein encoded by SV40 genome (the only one)
Hexamer = 6 subunits (2x3 copies of the same)
It s a helicase
Role = unwinds double helix at replication fork
What is the role of RPA?
Binds single-stranded + keeps it single-stranded DNA template in optimal conformation for DNA pol:
1. incorrect base pairing = higher energy (incorrect base=pairing is only 50x less favorable)
2. makes DNA pol move faster
3. keeps geometry of proteins s.t. wrong protein can’t access and form incorrect base pairing (error rate = 1/10,000 instead of 1/50)
Important for accuracy of DNA replication
What is the role of DNA Polymerase Epsilon?
Elongates leading strand by DNA synthesis
What is the role of PCNA protein in DNA replication?
Proliferating Cell Nuclear Antigen
It is a homotrimeric protein (forms a ring around template)
It prevents the Pol epsilon and delta complex from dissociating from the template
Forms a collar around DNA strands which also helps speed up replication
What are the roles of primase and of DNA polymerase alpha?
Primase forms RNA component of the primer
DNA polymerase alpha recongnizes RNA primer and extends primer with DNA
What is the role of the Replication Factor C (RFC) protein in DNA replication?
It is the PNCA loader, opens up the PCNA ring and loads it at a primer on DNA
What are the roles of Ribonuclease H and FEN-1 in DNA replication?
Ribonuclease H and FEN-1 displace (eats up) the RNA component at the 5’ ends of the Okazaki segment
What is the role of Pol delta in DNA replication?
it replaces RNA with DNA (in the Primer)
What is particular with the sequence of base pairs at origins of replications?
They tend to be AT-rich as it makes it easier to separate the strands
What are the general steps of DNA replication?
- 2 Helicase separate strands
- Unwinding (catalysed by large T-antigen (helicase) driven by hydrolysis of ATP + RPA binds and stabilizes single-stranded regions)
- Leading-strand primer synthesis (primase - Pol alpha complex)
- Extension (pol ε/ RFC/ PCNA complex replace primase-pol α complexes, extends the primer sequences)
- Further unwinding (binding of RPA to single-stranded regions)
- Further extension (pol ε/ RFC/ PCNA continue to synthesize leading strand)
- Lagging-strand primer synthesis (primase-pol α complexes form primers for lagging strand synthesis)
- Primer extension, primer removal and Strand Ligation of Lagging Strand
What is involved in the last step of DNA replication? (for the lagging-strand)
Primer extension, primer removal and Strand Ligation of Lagging strand:
1. Pol δ/Rfc/PCNA complexes replace primase-pol α complexes and extends the primer sequence
- Removal of RNA primers by FEN-1 and ribonuclease H
- Pol δ/Rfc/PCNA complexes replace the primer sequences with DNA
- Strands are ligated together by DNA ligase
How can DNA synthesis be qualified in terms of conservation of the already-existing information?
It is semiconservative
What are the 3 main reactions/steps of DNA replication?
Unwinding (helicase)
Polymerization (DNA pol)
Priming (primase)
How is DNA replication’s direction from replication origin?
It is bidirectional (a leading strand on the 5’ to 3’ end and a lagging-strand on the 3’ to 5’ end)
What reaction tend to change which acid base on DNA?
Purine gets hydrolysed (2000 - 10,000/day/cell)
Cytosine get deaminated (1/5days/cell)
Guanidine get Oxidized (1/5 days/ cell)
Adenine gets methylated (600/day/cell)
What is a mutation?
Permanent, transmissible change in genetic material
Can be spontaneous, by mutagens, by errors during replication
What is a mutagen?
Chemical compound, UV radiation or ionising radiation (x-rays, atomic particles) that increase frequency of mutations
Not all but many carcinogens are mutagens
Which are the different mechanisms of DNA repair ?
- Proofreading by DNA polymerase
- Base Excision repair
- Mismatch Excision repair
- Nucleotide Excision repair
- Double-strand breaks repair by end-joining
- Double-strand break repair by homologous recombination
Which DNA repair technique is responsible for the on the spot changes due to DNA polymerase’s errors?
go from 1/10,000 error, to 1/1,000,000,000 rate
Proofreading
Which DNA pol have proofreading activity?
epsilon and delta
3’ to 5’ exonuclease or “proofreading” activity
How does proofreading work?
Finger domain of DNA pol identifies incorporation of incorrect base → pol to stop
3- end of new strand moves freely to exonuclease domain site which digests the incorect base in new strand
Pol waits for correct new match to come in from the dNTP pool
What is the mose common point mutation (single base change)?
C to T
Deamination
-NH2 –> =O
Repair has to be identified directly (T-G) after deamination bc after replication, the base pair will become T-A and cannot be identified
*Repaired by Base Excision repairs T-G mismatches
How does base excision repairs T-G mismatch work?
*Want to replace T by C (to fit with G)
- DNA glycosylase breaks the H-bond between T and G + separate sugar phosphate backbone from T (only T leaves)
- APEI endonuclease cuts the strand where it is missing a base
- AP lyase (part of DNA Pol beta) removes deoxyribose phosphate
- DNA pol beta + DNA ligase uses G template to insert C, ligase repairs sugar-phosphate backbone (reforms 2 new phosphodiester bonds)
*Pol beta = replacement polymerase
How does mismatch excision repair work?
- MSH2 and MSH6 recognize the mismatch anc identifies which one is the newly synthgesized strand
- bind to the new daughter strand
- Trigger MLH1 endonuclease dimerized with PMS2. MLH1 cuts new strand (decides where to cut)
- DNA helicase unwinds and DNA exonuclease digests nucleotides of daughter strand
- DNA pol (epsilon) fills in the missing nucleotides using other strand as template + ligase repairs surgar-phsophate backbone
When is mismatch excision repair useful?
When replication is done as 2nd line of defence
Fixes base-pair mismatch, insertion, deletion of 1 or fes nucleotides
When does nucleotide excision repair occur?
Fixes DNA regions where chemically modified bases locally distort double helix
ex: Thymine-thymine dimer (adjacent T –> covalently bonded) caused by UV radiation
ex: Chemicals that bind to DNA bases
How does nucleotide excision repair work?
- Damage (distorted double helix) recognition by XP-C and 23B proteins
- Opening of DNA double helix: TFIIH (helicase activity) + XP-G + RPA (holds strands separated) unwind helix to make a 25 nuclotides bubble
- XP-F (at 3’ end) and XP-G (5’ end) cleave the phosphodiester bonds of the damages strand
- DNA pol fills missing nucleotides using other strand as template + ligase repairs sugar-phosphate backbone
Where does the name XP-n?
The endonucleases that cut phosphodiester bonds in Nucleotide excision repair
comes from xeroderma pigmentosum, a genetic disease that causes a high deposition to UV-induced cancer
Mutation that affect XP proteins cause this disease
What happens in the thymine-thymine dimer is not repaired and enters the replication fork?
- Normal replicative DNA pol (delta and epsilon) stall when reach dimer
- Pol ada (n) (special translesion pol) can read through but lacks proofreading activity which increases chances of mutation where the dimer was
- Eventually Pol n gets replaced by normal replicative DNA pol
What is the common point to double-strand Break Repair by End-joining and Double-strand Break Repair by Homologous recombination?
They repair clean breaks in on of both DNA strands (ex: clean separation of chromosome caused by RADIATION)
How does double-strand break repair end-joining (NHEJ) work?
Ku and DNA-PK binds the end 2 ends of double-strand break (DSB)
When 2 DSB bound by the complex come together, they recruit nuclease that remove several bases (to have blunt ends) –> deletion
2 double stranded molecules then ligated together
*This mechanism does not ensure the 2 belonging ends ligate together, can produce chromosomal rearrangement
What is the condition for double-strand break repair by homolgous recombination?
It has to happen in a diploid organism as it uses a homologous chromosome as template
What is the process of Double-strand break repair by homologous recombination?
- As replication process reaches a break in the phosphodiester backbone, replcation for collapses
- 5’-exonuclease digests few nuclotide from borken end (on the 5’-end side)
- top strand of the newly synthesized chromosome gets ligated to the top strand of the borken chromosome (according to template from bottom strand of broken that is sticking out after having digested part of the top strand)
- RecA- or Rad51- mediated strand invasion:
What is BRAC1 and BRAC2?
human gene that plays critical role in stability and integrity of DNA
BRAC1 and BRAC2 deficiency → breast cancer, ovarian cancer
Important role in repair of double-strand breaks in DNA by homologous recombination
What is hereditary nonpolyposis colorectal cancer?
What system does it affect?
Disease that also affects DNA mismatch repair
Caused by UV irradiation, chemical mutagens
Early developement of tumors → Colon, ovary cancer
What is Xeroderma pigmentosum?
What system does it affect?
Affect Nucleotide excision repair
Sensitivity: UV irradiation, point mutations
Symptoms: Skin and eye photosensitivity, keratoses
Cancer: Skin, carcinomas, melanomas
What is Bloom’s syndrome?
What system does it affect?
Affects repair of double-strand breaks by homologous recombination
Sensitivity: Mild alkylating agents
Cancer: Carcinomas, leukemias, lymphomas
Symptoms: photosensitivity, facial telangiectasis, chromosomes alterations
What is Fanconi anemia?
What system does it affect?
Affects repair of double-strand break by homologous recombination
Sensivity: DNA cross-linking agents, reactive oxidant chemicals
Cancer: Acute myeloid leukaemia, squamous-cell carcinomas
Symptoms: Developmental abnormalities (infertility and deformities of the skeleton), anemia
What is topoisomerase?
It is an enzyme involved in DNA replication that relieves supercoils to be able to unwind DNA afterwards
So DNA doesn’t get all tangled up
