DNA Replication & Repair Flashcards
What is the major difference between DNA replication on the leading and lagging strand?
Leading strand is continuous
Lagging strand is discontinuous
Define semi-conservative in the context of DNA synthesis
each strand of double stranded DNA serves as template for synthesis of new strand -> when 2 new molecules of DNA are formed each has 1 NEW & 1 OLD STRAND
Define bidirectional in the context of DNA synthesis
Replication is seen at both sides of replication fork (always 5’ to 3’ but goes in opposite directions- relatively- on leading and lagging strands)
Describe Okazaki fragments
fragments of discontinuous lagging strand synthesized in 5’ to 3’ direction, OPPOSITE OF FORK MOVEMENT
- few hundred to few thousand bp long
- synthesis stops when it gets to RNA primer
Describe the origin in the context of DNA synthesis
unique point where replication loops always begin, in circular DNA the 2 replication forks ultimately meet opposite origin
Describe the replication fork
Point where parent DNA is being unwound and separated strands are quickly replicated
What is the function of the origin binding protein?
Recognize the replication origin
What is the function of a helicase?
Melt/ unwind DNA
What is the function of single stranded binding proteins (SSB)?
Protect/ stabilize unwound DNA strands (single stranded DNA)
What is the function of primase?
responsible for synthesis of RNA primer, primosome constituent
What is the function of DNA polymerase I?
Fills gaps
excises primers
removes RNA primer
Requires template & RNA primer
What is the function of DNA polymerase III?
Elongate DNA from RNA primer
What is the function of DNA ligase?
Ligates or connects DNA fragments
i. e. Okazaki fragments
- ATP as cofactor in Eukaryotes
- NAD+ as cofactor in prokaryotes
What is the function of the sliding clamp?
Pol I is inefficient in synthesis because it goes on and off
Pol II associates with sliding protein that clamps on to DNA strand and prevents disassociation
Increases efficiency of synthesis
Increases processivity of DNA polymerase
PCNA is sliding clamp in Eukaryotic DNA
Pol gamma-beta subunit is sliding clamp of prokaryotic DNA Pol III
What is the function of Topisomerase/ gyrase?
Relieve torsional strain in front of replication fork - breaking and rejoining double stranded DNA
Prevents over-winding
What is the function of telomerase?
adds telomeres to stabilize DNA
What is the function of reverse transcriptase?
RNA dependent DNA polymerase (DNA from RNA)
Describe how DNA polymerase creates phosphodiester bond during addition of deoxyribonucleotides (dNTPs)
DNA pol I requires 3’ -OH on growing strand to act as primer and attack phosphoanhydride bond on next nucleotide
New nucleotide is added here with 3’ -OH primer for next bp addition
Addition based on pairing with template (non-coding) strand
=> dNMP (DNA) + dNTP -> dNMP (lenthend DNA) + PPi
What is the function of exonuclease?
Proofread DNA polymerase and correct errors
Describe the order of events that occur during, differences between, and coordination of DNA synthesis on leading and lagging strands
Leading strand ->
- Continuous copying following RNA primer after addition of ORC, follows helicase and gyrase/topoisomerase
- Moves in same direction as replication fork
Lagging Strand ->
- Uses RNA primer from previous okazaki fragment -> synthesizes 5’ to 3’ -> segments of varying lengths -> connected by ligases
- Discontinuous copying
- End replication problem – lagging strand cannot be synthesized to end during DNA replication (telomeres continuously shorten)
- Need RNA primer to begin synthesis of each piece of lagging strand DNA- at end there is no place for this to attach
- => ultimately telomeres are short enough that they initiate signal for cell death (apoptosis)
- In cancer telomerase enzyme de-repressed – restores telomeres – blocks normal cell death of old cell
Why is DNA repair so important?
- Damaged RNA quickly digested-> low impact
- DNA is irreplaceable => imperative to maintain
- Change in DNA=> mutation (permanent change to bp sequence) Mutation
- Often results in cancer
- DNA is only repaired macromolecule
How does DNA damage occur?
During replication From products of normal cell metabolism Radiation Chemicals (alkylation) Loss (depurination, depyrimidation, deamination) UV exposure
What are the 3 types of mutation?
Point mutation
Insertion
Deletion
What specific type of DNA damage does UV exposure cause?
Thymidine dimer
What permanent mutation can deamination of cytosine result in?
Deamination of cytosine results in uracil
If not repaired, A incorperated rather that G, permanently mutate to AT instead of GC
Explain mismatch repair
removes misincorperated nucleotides during DNA replication (distinguishes between template and new strand)
- Recognize damage/ mismatch
- endonucleases mediated cutting of phosphodiester backbone on either side of error
- Nuclease mediated removal of DNA fragment with error
- DNA polymerase med synthesis of missing nucleotides from intact strand
- DNA ligase med sealing of nick in backbone
- Initiated by 2 protein complexes -> MutS (hMSH) & MutL (hMLH & PMS)
- Differentiates between old and new by degree of methylation
- Repairs mismatches (point mutation)
Explain base excision repair
repairs DNA lesions missed by nucleotide excision repair
- Geared towards thing like Deamination caused errors
- Requires glycosylase family enzymes – each recognizes specific base
- Glycosylase “flips” altered base out producing empty space
- AP site removed by AP- specific endonucleases & AP lyase
- DNA polymerase fills nick
Explain nucleotide excision repair
Removes damage that distorts DNA structure and block polymerase function
- Recognition and binding of damaged site by multi-protein complex
- Local unwinding of DNA by helicase (TFIIH protein complex) -> ~25bp bubble
- Double incision of damaged strand and removal of ~30bp w/ lesion
- DNA polymerase fills gap
- DNA ligase joins ends
- Geared towards things like : thymine dimmers & bulky DNA adducts
Explain homologous recombination
-Repairs DS break
-Requires sequence homology between broken ends and template DNA
-Very accurate
Only occurs during S and G2 phases
Explain non homologous end joining
-No sequence homology
-Repairs DS break
-Often leads to insertion or deletion
Quick and dirty
Explain direct reversal repair
- ligation of break in phosphodiester backbone by DNA ligase
- Repair of O6-methylguanosine by O6 methyguanosine methyl transferase
What mechanism allows DNA replication to continue in face of DNA lesions that other repair pathways fail to remove?
DNA damage tolerance/ bypass: trans lesion synthesis
What is the unfortunate consequence of DNA damage tolerance/bypass?
Error rates 100 to 10,000 times higher than normal
How does DNA damage tolerance/ bypass: trans lesion synthesis work?
- If cells have too much damage for NER, BER, MMR repairs, esp replication blocking lesions
- Bypass is last resort – allows loosened specificity ( loook away)
- Lacks 3’ to 5’ exonuclease activity
What is the role of DNA damage checkpoint in maintaining gnome stability?
Cellular “surveillance” mechanism
- Arrests cell progress through cell cycle to allow for repair of DNA
- Includes: damage sensors, signal transducers, effectors
- ATM & ATR=> lesion detection (then recruit downstream proteins)
- ATM & ATR at high levels in cells in early stages of tumorgenesis
- Mutations affecting DNA damage checkpoint=> genomic instability, malignant transformation
Why is looping at the 2 core proteins important?
It allows concurrent synthesis of leading and lagging strand in 5’ to 3’ direction (okazaki fragments loop back and go through core protein in same direction as leading strand)
Allows DNA to use single complex to synthesize both leading and lagging strand
What is the error rate of DNA polymerase?
1 incorrect base pair per 10^4 or 10^5 correct base pairs
What is the error rate after proof reading acitivity of DNA polymerase?
1 incorrect base parir per 10^6 or 10^8 correct base pairs
