dna replication and repair Flashcards
unwinding of the two strands , exposed bases , and strict Watsons-crick base pairing ( becoming the complementary strand as the double strand of dna unwinds )
semiconservative replication ( aka one parental strand and one newly seznsthesied )
dna replication requirement:
1- single strand template
2- deoxyribonucleotide triphosphate ( dNTPs of A,G,C,T ) + Mg +2
3- replisome: nucleotide protein complex that co-ordinates the replication activities numerous enzymes and proteins
4- primer w/ a free 3’ end hydroxyl group
—- is the separation of 2 complimentary strands that occurs in the origin of replication
initiation
true or false:
1- specific points where dna replication begins consensus sequence - short AT poor region
2- eukaryotes have one site of replication
1- false: rich
2- false: mutible site
from the origin 2 —- move outwards in — direction.
Active systhesis requires the assembly of — at the origin of replication
- replication forks
- opposite
- replisomes
what are the roles of protein in replisomes?
1st step : unwinding proteins
- dna helicase ( separates the double strands into a single strand to allow the dna to be copied )
- single strand binding protein SSB ( wraps the single strand of dna )
- topoisomerase ( which prevents dna from getting tangled , can cut the backbone and reform it )
2nd step : enzymes to replicate
- primase ( sytheises the short rna sequence that is complementary to the single strand of dna that serves as a template )
- dna polymerase ( sythezies the dna , works in one direction , only adds to the 3’ , copies info from the template strand to make the daughter strand )
in the dna polymerase it uses — strand of the dna as a template , it reads its template from —– and it make new dna from —-. It aligns and adds nucleotides alone the ss template which specifics the —- of the new chain aka —— , catalysis formation of —–
- single
- 3-5
-5’ - 3’ - sequence
- Watson crick base pairing
- phosphor bonds
(DNA)n + dNTP —> (DNA)n+1 + pyrophosphate ( PPi ) this reaction is driven by the subsequent hydrolysis of PPi
dna polyemrase is highly — < or equal to 1000 base/second such as: —– and —– , and dna polymerase has — activity to prevent errors such as:
- highly processive as
1- PCNA proliferating cell nuclear antigen
2- sliding camp role: it encircles the dna template and keep the dna polymerase closely associated to template as it rapidly move along , the catalyses bond formation joining < or equal to 1000base / second - it has proofreading activity as:
1- substrate specificity
2- proofreading
for the dna polymerase substrate activity its active sure can bind to all —- snf catalysis occurs when the — one is bound . The dNTP base pairs with template while the enzyme is in — and catalytically — form . the enzyme conformational change with the correct W-C pair leading to —-
- all four dNTP types
- correct
- open
- inactive
- active enzyme
3’-5’ exonuclease activity acts as —- in reverse direction as it removes the nucleotide at the — end of the new strand that are mismatched
- proofreading error correction activities
- 3’ end
the leading strand is synthesised — by the — travelling the replication form since the dna polymerase makes new copies
from 5’-3’. While the —- is sytheised discountoisly piece by piece which is what we call —-
- continuously
- dna polymerase
- lagging strand
- semidiscointous replciaropn
the lagging strand is sytheises piece by piece as the primase make a new — at the regular intervals , dna polymerase replicates the template from the primer producing a new strand in —- direction , dna is blocked by proxmity of the next primer and the result is a dna strand of 1,000bp —— , primer is — , — filled and — joined
( check slide 14 for pic)
- primer
- 5-3
- Okazaki fragments
- removed
- gaps
- backbone
in eukaryotic dna polymerase the human have multible enzymes and the main ones are:
poly alpha ,delta , and epsilon
—- is involved in initiating the replication and is associated tightly w/ primase to make a —-
poly alpha , poly alpha / primase complex that is 7-10 rna + 15dNTPs
poly alpha replicated the dna by —– 5’-3’ and it has —- and the processive is —-
- extending the primer
- no exonulceas activity aka no proofreading
- moderate
poly delta and epsilon are not associated with — and they replicate the dna by extending the primer 5-3 and their processive is — , they have — in complex with —- and they have —- activity
- primase
- high
- unlimited in complex w/ PCNA
- 3-5 exonuclease activity
— is responsible for the leading stranding synthesis
—- is responsible for the lagging strand sythesis
- poly epsilon
- poly delta
the two enzymes required for the primer removal are:
1- Rnase H1
2- Flap endoculease 1 ( FEN1 )
( check slide 17)
— removed most of the rna leaving one 5- ribonuclide adjacent to the dna
—- removes the 5- ribonucleotide which has the endonuclease activity and poly alpha lacks proofreading , and this has endoculeas activity but the mismatch is up to — from 5- end of annealed dna
- Rnase H1
- FEN1
- 15 bp
eukaryotes lack — sequence , dna replication proceeds until each replication fork — with the fork from the adjacent replicon
the problem w/ replicating the 2 ends of linear dna strands called —- , the continuous synthesis on the leading strand can proceed to very tip of the template. but what happenes to the extreme end of the lagging strand????
- primer is removed but the — available for the dna polymerase to add nucleotide so the dna can be replicated at the end of the strand ( that’s for the leading strand unlike the lagging strand it doesnt have that )
- termination
- collides
- tolemeres
- OH
3’ end of each chromosome —- of the tandem repeats which is — in humans
- 1,000
- TTAGGG
telemetric dna synthesised and maintained by — = —–
the – acts as a template for the sytheiss of the dna and adds tandem repeat to the — end
- telomerase = ribonucleoprotein ie. rna + protein
- rna
- 3’
the normal tolmerase activity is —- the cells as in unicellular eukaryotes and in humans as ——- production
1- dividing
2- germ line and gametes production as sperm
during the development as cells divide and differentiate the telomerase function —–
declines = tolermease shortens
telomerase contain approx –kb hexamer repeat after 100s of cell division :
the chromosome end will be — and gene will be —- which causes the dna to be —- causing the cells to stop dividing and tier g0 or apoptosis
- 16kb
- damaged
- deleted
- damaged
—— maintain chromosomal stability and prevent chromosomal degradation
tolemere
Some cells have the ability to reverse telomere shortening by expressing —–
tolemerase
—– is an RNA-dependent DNA polymerase, meaning an enzyme that can make DNA using RNA as a template.
tolemerase
—- shorten each time the cell replicates until they can no longer divide which leads to —- , however the enzyme — takes place to restore the cell division however it can cause —-
- telomere
- aging
- tolemrase
- cancer
the absence of —- allows normal senescent of somatic cells aka aging , while the abnormal activity ( enhanced activity ) results in —-
telomerase
uncontrolled division leading to cancer , diagnosis tool potential target for treatment
true or false:
the proofreading activity of dna polymerase reduces the error rate by 1 in 10 power 4 - 10 power 5 to 1 in 10 power 7 base replicated
true
dna is constantly being damaged by :
radiation , high energy radiation , chemicals
the damage is the uv light which allows the fuse of adjacent pyrimidines
radiation
the damage causes the double strand to break
high energy radiation
the damage affects the nitrous acid by delaminating the amines
chemicals eg. c –> uricle , a—> hypoxanthine
most damage is repaired by the —- while the remains can cause: —— and —- in the dna sequence
these damages can be:
- cells
- mutation and changes
- insertion , deletion and base substitution
—– occurs shortly after replication , replaces mistamche bases or loops up to 4bp in the dna
defects in humans may cause —-
mistmatch repair MMR , high cancer incidents
methylation aka the discrimination between parental and daughter strand is an example in
mismatch repair
HNPC ( heredity non polyposis cancer ) 70% occurs in —– and —- genes and produce a —- protein and this occurs in — repair
- MLH1 + MLH2
- MutL proteins
- mistmatch
—- replaces bases lost through chemical process such as deamination and depurination
base excision repair
( includes: dna glycolase , ap endocunxlease and exonuclease )(dna polymerase and ligase for repair)
—- identifies and removes damaged base leaving apurinic or apyridindic sites
—- cuts the backbone
—- removes the sugar + several adjacent bases
dna glycosylase
AP endonuclease
exonuclease
—- responds to helix distortion as pyrnimidne dimers , replaces regions of damaged dna of up to 30 bases in length , defence against 2 important carcinogens ( tabacco smoke and sunlight )
nucleotide excision repair
( read:Humans: 16 proteins
Mutations affecting different proteins in the
pathway identified in two disorders:
Cockayne Syndrome microcephaly, premature
aging, sensitivity to sunlight, developmental
delays, shortened lifespan.
Xeroderma Pigmentosum)
—– is a rare human skin disease –
Autosomal recessive
Deficiency in nucleotide excision repair;
lack of enzymes necessary for repair of
DNA damage induced by ultraviolet
(UV) radiation (Thymine dimers)
Symptoms:
- extreme sensitivity to light
- skin cancer
- frequent secondary tumours & associated cancer-related
death (< 30 yrs. of age)
Xeroderma pigmentosum ( XP)
mechanism for repairing the double strand break - nonhomologous end-joining NHEJ :
The “Ku protein” = a broken DNA
sensor, recognises double stranded
breaks.
The Ku protein holds both strands of
broken DNA leaving the ends
accessible to: nucleases
polymerases
ligases
Ends of broken DNA aligned,
trimmed or filled and strands ligated
Generates mutations
Deficiency - associated with
predisposition to cancer and
immunodeficiency syndromes
mechanism for repairing the double strand break:
( recombination or homologous repair )
Uses enzymes and proteins that perform
genetic recombination between homologous
chromosomes during meiosis
Uses DNA sequence information in
homologous chromosome to correct the break
During S phase, sister chromatid is physically
close, providing a homology donor for repair
Non-mutagenic
Important in humans :
Defects in proteins BRCA1 and BRCA2
incidence of breast, ovarian, prostate,
pancreatic cancers
Mutation in genes coding for BRCA1 and BRCA2
= 80% lifetime risk
