DNA Replication and Repair Flashcards
Exam 2
Explain the steps of DNA replication and neccesary biomolecules for each step.
Overview:
* DNA replication is an essential process for every life form. Before cell division, chromosomal DNA needs to be replicated to make another copy of the genetic information.
* DNA replication is very fast! The himan genome consists of 6 billion base pairs. The replication of the human genome takes about 8 hours. 200,000 bp per second due to multiple relication origins (40,000-80,000)–> super accurate
* Super accurate! An error during replication results in a heritable mutation, which may cause a genetic disease or cancer (constant). During a single cycle of replication, one error per one billion pase pairs occurs. More mutations are generated by chemicals and radiation that replication errors. DNA repair is neccesary.
Steps defined by biomolecules:
DNA Polymerases
* Synthesize DNA by adding a nucleotide complementary to the template DNA; the genetic information is conserved.
* Requires the 3’ OH group of a pre-existing primer; synthesis occurs in the 5’ to 3’ direction while moving on the template in 3’ to 5’ direction.
* Human cells have multiple DNA polumerases (alpha, beta, gamma, delta, etc.) with various roles; DNA polymerase delta is the major enzyme for DNA replication
dNTPs
* Deoxyribonucleotides (dATP, dTTP, dCTP, and dGTP)
* Substrates for DNA synthesis
* DNA replication stops when dNTPs are not sufficient–> cell cycle arrest!
* We do not use paritally synthesized DNA, if we do cells die.
Formation of the phosphodiester bonds:
* 3’-OH group makes a phosphoester bond with alpha-phosphate of dNTPS
* The resulting pyrophosphate (ppi) is released.
Facts:
* It takes 8 hours to copy the entire DNA polymerase
* Need 6 billion ATP for the entire process
Error–> mutation –> cancer/disease
Identify the components of the DNA replication fork.
Leading Strand:
* New template sequence appears in front of the synthesis
Lagging Strand
* New template sequence appears behind the synthesis
* DNA synthesis is discontinuous, producing Okazaki gragments
* Okazaki fragments need to be Linked by Ligase
Polymerases working on the leading and lagging strands form a large complex called replisome (area where replication occurs).
Describe how cells maximize the accuracy and speed of replication.
Accuracy
Proofreading
* DNA polymerase delta has the 3’ to 5’ exonuclease activity, which removes the newly-added nucleotide.
* The nucleotide at 3’ end dictates the relative tendency of synthesis to proofreading.
* A proper nucleotide suppresses proofreading
* A mismatched nucleotide suppresses synthesis.
* Critical for the accuracy of DNA replication.
* Balance between efficency and accuracy is necessary
* DNA polymerase alpha does not have proofreading ability.
Speed of Replication
Processivity
* DNA polymerases may dissocciate from DNA before the completion of synthesis. Rebinding of a DNA polymerase slows down the replication.
* Enhanced processivity increases the rate of DNA syntheses–> processivity= stays longer to continue “processing”–> high staying=high processivity
* Sliding clamp
* In human cells, PCNA (a sliding clamp) holds down DNA polymerase delta and supresses its dissociation from DNA. This increases processivity significantly, which means faster synthesis.
Explain how cells lose or extend the telomere sequence.
Basics of Telomeres
* Help end DNA replication procrdd
* Human chromosomal DNAs are linear
* A portion of 5’ end of the lagging strand is not replicated because a primer cannot be made
* The chromosomal DNAs are shortened by 50-100 base pairs per replication
* Telomeres are located at the ends of chromosomal DNAs.
* Contains thousangs of the repeat TTAGGG without any genetic information
* Lost after 40-60 replications–> cell death (Hayflick limit) –> cancer cells shorten telomeres to kill cancer cells.–> loss of telomeres ends the process of DNA replication–> we are as limited as our telomeres.
Telomerase in Action
* Ribonucleoprotein complex; contains RNA
* Adds TTAGGG to the 3’ end of telomere using the RNA sequence as the template.
* This elongates the telomere and increases the lifespan of cells.
* IF telomere doesn’t get lost after 40-60 replications, the cell will be immortal and keep dividing forever
Describe the step of polymerase chain reaction and the neccesary materials for the reaction.
What is a polymerase chain reaction?
* Amplifies a DNA fragment of interest defined by the DNA primers in vitro.
Steps: denaturation, annealing, and extension
- Requires a thermocycler and heat-resistant polymerase like Taq of Pfu from thermophilic bacteria. This is because the polymerase functions at high temperatures due to needing high temperatures to denature DNA.
- The cycle is repeated 25-30 times
- Commonly used for cloning and detection
dsDNA–> denaturation—> DNA primers bind to specific sequence—> lower temperature—> annealing—> High temp—> extension or original DNA
List the various causes of DNA damages.
Internal Causes
1. Reactive oxygen species like HO or superoxide. ~20,000 8 oxy-guanine in human cells a day
2. The deamination of cytosine to uracil and adenone to hypoxanthine. C2T mutation occurs to change cytosine to uracil via deamination.
3. Depurination–> Several thousands of purines are lost in human cells in a day. Generates abasic AP sites, which are gaps because the base is gone!
External Causes
1. Carcinogens–> mustard gas (methylation of DNA), N-nitrosos compounds in cured meat (introduces erro in normal cells), and chemotherapy (we use carcinogens to kill carcinogens–> if chemo too frequent we give cancer to healthy cells).
2. UV Radiation (200-300 nm)–> pyrimidine dimers (cytosine and thymine). Thymine dimers are more frequent then others. Not fixed=mutations.
Describe the cellular responses to different types of DNA damages or errors.
DNA Repair
Base-excision repair (BER)
* DNA glycosylases remove damaged bases–> abasic (AP) site is formed.
* AP endonuclease and exonucleases remove the AP site by cutting some DNA and resynthesizing it to seal the gap
* DNA polymerases and ligases resynthesize and seal the gap.
Nucleotide-excision repair (NER)
* A stretch of nucleotides with a damage (~30 residues) is removed, and the gap is resynthesized.
* At least 16 proteins are involved; discovered mostly by the mutations in genetic diseases, such as XP.
* Major defense against Sunlight and tobacco.
Xerodoma pigmentosum (XP)
* Autosomal recessive genetic disease
* Defect in the nucleotide-excision repair system
* Photosensitive and susceptive to skin cancer (~2000-fold greater thean the normal population).
* We get the same DNA as the pt when the sun hists us, but we have the defense against it (NER). However, with XP you are WAY more likely to die from skin cancer.
Mismatch repair (MMR)
* Fixes a mismatched but undamaged base pair.
* Similar to the nucleotide-excision repair.
* If the correct strand is removed, a mutation occurs.
* Methylation of DNA may guide the identification of the parental strand.
Direct Methylation
* Alkyltransferases remove the methyl group from O6-methylguanine.
Photoreactivation
* DNA photolyases revert pyrimidine dimer
Double-strand break repair
* ~10 double strand breaks occur during a single cell cycle
* Homologous recombination- accurate bc we don’t lose any
* Non-homologous end-joining- error prone
Explain the role of p53 in DNA repair.
- Master controller of DNA repair
- transcription factor responding to DNA damages
- “The guardian of the genome”
Functions:
* Activates DNA repair proteins
* Arrests cell cycle
* May initiate programmed cell death if the DNA damage is overwhelming.
Tumor suppressor:
* Failure of programmed cell death may cause a cancer
* Frequently mutated in human cancer (>50%)
p53= the protein that induces the cell cycle
If it is broken, the cell can become cancerous.
p53 not broken= major cancer supressor
Explain the replication process
I am in my replication era
- A helicase (MCM) unwinds dsDNA. Newly exposed single stranded DNA are stabilized by replication protein A (RPA)
- A primase synthesizes a short RNA primer (single stranded)
- DNA polymerase synthesizes DNA in the 5’ to 3’ direction. Started by polumerase alpha, which is replaced by DNA polymerase delta.
- The RNA primers are digested by RNase H. The Flap endonuclease-I (FENI) removes any mismatches introduces by DNA polymerase alpha.
- DNA polymerase delta synthesize DNA to the end of the Okazaki fragment in front.
- A ligase seals the nick in the DNA strand.
Disclaimers:
1. RNA primer focuses on ONE strand, which is error prone (don’t want to introduce it)
