DNA Replication in Eukaryotes Flashcards
What is the eukaryotic version of helicase?
MCM
What is one difference about the replication fork/complex in eukaryotes?
A pre-replication complex (pre-RC) must form on ARS (origin of rep) before helicase can act
More Pre-RC are ______ (assembled) than _______ (used).
Licensed
Fired
What causes Meier-Gorlin Syndrome?
Mutations in genes that encode the proteins required for the the Pre-RC complex.
What are symptoms of Meier-Gorlin syndrome?
Failture to thrive
Patella hypoplasia
Small upper and lower jaw
What causes DK?
Defective telomerase/telomere shortening
What is unique about telomerase?
It contains a reverse transcriptase component
Why are telomeres needed?
The lagging strand gets left with a left over primer that when removed, would shorten the DNA. Telomerase replaces the gap.
How does telomerase work?
It positions itself at the end, add a telomere repeat, reposition, etc.
Do telomerases use DNA or RNA?
RNA
In order to protect itself telomeres…
Loop back on themselves and create a t-loop and a d-loop
What are symptoms of DK?
Premature aging Oral leukoplakia Skin hyperpigmentation Nail dystrophy/ridging Bone marrow failure/aplastic anemia
What is Hoyeraal-Hreiderasson (H-H) syndrome?
Rare variant of DK
Diagnosed in first months
What are the symptoms of H-H syndrome?
Aplastic anemia
Severe immunodeficiency
Cerebellar hypoplasia
Mutation in hTERT, hTR
Disease of telomere shortening show anticipation due to loss of telomerase function in _____ cells.
Germ
What are the two major methylated bases in prokaryotes?
Adenine and Cytosine:
N6-methyladenine
N4-methylcytosine
What is the role of methylation in bacteria?
Protects bacteria’s DNA from cleavage by restriction endonucleases
In E. coli methylation of adenine residues in the sequence ____ is involved in mismatch error correction.
GATC
What bases are methylated in eukaryotes?
Cytosine only!
5-methylcytosine
What is the sequence of methylation in eukaryotes?
CG and complementary CG will be also methylated in the corresponding position
Is methylation heritable or inheritable?
Heritable
A ______ promoter tends to be expressed and a _______ promoter tends not to be expressed.
unmethylated = expressed
methylated = not expressed
How can you reverse the methylation of genes?
Treatment with 5-azacytidine, a cytidine analog that cannot be methylated
What are common causes of DNA mutation?
Mistakes during replication
Deamination of cytosine
UV damage
Chemical damage
What happens due to deamination of 5-methylcytosine?
CG to AT transition mutation
What is a transition point mutation?
Exchange of one pyrimidine-purine base pair for another pyrimidine-purine pair.
What is a transversion point mutation?
Pu-Py exchanged for Py-Pu
This is worse!
What are common causes of deletion or insertion mutations?
Intercalating agents that fit in between adjacent base pairs
Transposable elements
Triplet repeats
What is the worst result of a deletion/insertion mutation?
Changing the reading frame
What are inter or intra strand break and crosslinks?
Photodimerization for example causes intra stand dimerization of adjacent (pyrimidine) dimers.
Is O6-alkylguanine an especially deleterious form of DNA damage?
Yes
What does O6-alkylguanine have a high probability of being base paired with during replication?
Thymine
How are O6-alkylguanine repaired in prokaryotes and eukaryotes?
O6-alkylguanine DNA methyltransferase (MGMT) transfers the methyl group to itself i.e. self alkylates and restores the guanine.
How does excision repair work?
Damaged DNA is recognized, removed, and then replaced by DNA polymerase
What does nucleotide excision repair (NER) fix?
Intra strand thymine dimers caused by UV radiation
What are the steps of NER?
- Recognition of damage
- Repair proteins stall at damage
- Bend DNA
- UVrA displaced, UVrC joins complex
- Endonuclease cuts 3’ to 5’ and a helicase removes damaged piece
- DNA Pol I replaces excised DNA
- DNA ligase seals nick.
What is base excision repair?
DNA-N-glycosylases removed incorrect BASES in DNA
What enzyme removed mismatched uracil in prokaryotes?
Uracil DNA-N-glycosylase
What enzyme repairs DNA in eukaryotes?
DNA Pol Beta
What two types of mechanisms exist for repairing eukaryotic DNA by excision repair?
Short patch BER
Long patch BER
What is mismatch repair?
Nascent DNA scanned for errors - mismatched bases and single base insertions or deletion
Errors are corrected
How does mismatch repair occur?
MutHLS system:
- Recognition of damage
- MutHLS complex searches for methylated GATC sequence closest to damage
- MutH (endonuclease) cuts the backbone 5’ to the G in GATC
- Helicase unwinds DNA past damage
- Exonuclease removes DNA past damage
- DNA Pol III fills the gap
- Nick in backbone sealed by DNA ligase
It looks for methylated strand because it knows that is the correct parent strand. Daughter strand not yet methylated
What genes are affected in patients with CMMRD?
MSH (mismatch repair) genes
What are common symptoms of CMMRD?
Childhood onset of brain tumors Colorectal cancers Neurofibromatosis-1 Cafe au lait spots Hematological malignancies
What are symtpoms of xeroderma pigmentosum?
Superficial reddening of the skin
Freckling
Rapid development of skin cancer despite little sun exposure
What causes xeroderma pigmentosum?
Failure to conduct nucleotide excision repair
What are the two types of eukaryotic NER?
Global genome NER: probes the genome for lesions
Transcription-coupled NER: removes transcription-blocking lesions to permit unperturbed genome expression
