Lecture 3: DNA Replication, Repair, and Recombination 1 Flashcards
What is the germ-line error rate (mutation rate) in humans?
- About 70 new single-nucleotide mutations in the offspring’s germline when compared to parental germline.
- Mutation rate of one nucleotide change per 1x10^8 nucleotides per generation.
How are most germ-line errors corrected?
-
MOST errors are corrected by:
- proofreading (polymerase activity)
- DNA repair
- Errors are further corrected by post-replication repair mechanisms.
Why is it so crucial for multicellular organisms to have high fidelity replication?
- Germ cells have to have low mutation rates to maintain the species.
- Somatic cells need low mutation rates to avoid uncontrolled proliferation/cancer.
What is the catalyzing reaction by DNA polymerase when synthesizing DNA?
(DNA)n+1 residues + dNTP -> (DNA)n+1 + P2074-
What does DNA polymerase require to begin?
Requires a primer with a free 3’-OH to begin.
What is released from the reaction when DNA polymerase addes a new deoxyribonucleoside triphosphate to the growing strand during replication?
Pyrophosphate
True or False:
DNA polymerase can only synthesize DNA in the 5’-to-3’ direction.
True
True or False:
The replication fork is symmetric.
False - replication fork is asymmetric.
Between the leading and lagging strand, which one is synthesized continuously and which one is synthesized in segments?
- Leading Strand = synthesized continuously
- Lagging Strand = synthesized in segments
What fragment is produced in segments during lagging strand synthesis?
Okazaki Fragments
How often does DNA polymerase make a mistake?
Every 1x109 nucleotides copied.
How are the mistakes that DNA polmerase make sensed by the replication machinery and fixed?
- Before a new nucleotide is added the enzyme must tighten its “fingers” around the active site, which is easier if the correct base is in place.
- If incorrect base - this is sensed by the palm not being able to close down correctly, which trigger the proofreading (exonuclease) activity of DNA polymerase.
- DNA polymerase has exonuclease activity in the 3’-to-5’ direction that will fix the incorrect base.
When does exonucleolytic proofreading take place?
Immediately after incorrect base is added.
During exonucleolytic proofreading, what does the exonuclease do if the base is incorrect?
- 3’-to-5’ exonuclease clips off unpaired residues at 3’ primer terminus.
- DNA polymerase requires a perfectly paired 3’ terminus to continue replication.
Why is it important that replication proceeds in the 5’-to-3’ direction?
- 5’-to-3’ replication allows efficient error correction.
- in a hypothetical 3’-to-5’ strand growth, if proofreading takes off a nucleotide then the reaction does not proceed, as no high-energy bond would be cleaved (thus, not favored by the cell).
- in 5’-to-3’ strand growth, if proofreading takes off a nucleotide then a high-energy bond is cleaved, providing the energy for polymerization (thus, favored by cell).
What type of process is the lagging strand replicated through?
- Backstitching Process:
- DNA primase synthesizes an 10 nt long RNA primer to prime DNA synthesis.
- DNA polymerase can’t initiate de novo synthesis - this would increase the mutation rate.
- RNA primer is erased by RNAseH (recognizes RNA/DNA hybrids) and replaced with DNA; DNA ligase joins the ends.
- DNA primase synthesizes an 10 nt long RNA primer to prime DNA synthesis.
You know that inorder to prime DNA synthesis DNA primase must synthesize a 10 nt long RNA primer. What enzyme erases the RNA primer?
RNAseH (recognizes RNA/DNA hybrid) and replaces it with DNA; DNA ligase joins the ends.
At what rate can DNA helicase unwind DNA?
Capable of prying apart the helix at rates of 1000 nucleotides pairs/sec.
How many subunits make up DNA helicase?
- Protein with 6 identical subunits that binds and hydrolyzes ATP.
- this causes conformational change that propels it like a rotary engine along single stranded DNA, passing it through a center hole.
What is the function of single-stranded DNA binding proteins (SSB)?
- Bind tightly and cooperatively to exposed SS DNA.
- help stabilize unwound DNA
- prevent formation of hairpins
- DNA bases remain exposed
**bind to keep the single stranded DNA so the DNA polymerase can stay on and correctly replicate long stretches**
What is the function of the sliding clamp?
- Keeps DNA polymerase on DNA when moving.
- releases when double stranded DNA is encountered
What is the function of the clamp loader?
(hint: assembly requires clamp loader)
Hydrolyzes ATP as it loads the clamp onto a primer-template junction.
True or False:
On the leading strand the clamp remains associated to DNA polymerase for long stretches.
True
True or False:
On the lagging strand the clamp loader stays close so it can assemble a new clamp at the start of each new Okazaki fragment.
True
What does mismatch repair do?
Removes (almost all) errors missed by proofreading by detecting distortion caused by mispairing.
How does mismatch repair know which strand is correct?
- In E. coli, depends on methylation to distinguish new strand.
- MutS binds to mismatch
- MutL scans for the nick and triggers degradation of nicked strand
- In humans, depends on single strand breaks.
- present on lagging strand before Okazaki fragments are ligated.
- leading strand not known
What happens if there is mutations in the mismatch repair gene?
- cells accumulate mutations at high rate
- HNPCC
- Hereditary Nonpolyposis Colorectal Cancer
Look over slide 17 - 2015 Nobel Prize in Chemistry
Given to three sicentists for “mechanistic studies of DNA repair”.
What is the function of DNA topoisomerases?
- As replication fork moves, it creates a winding problem for the parental helix.
- Every 10 bp replicated corresponds to one turn.
- DNA topoisomerase is a reversible enzyme that breaks a phosphodiester bond to change superhelicity, thereby relieving supercoiling.
What is the function of type I topoisomerase?
- Catalyze the relaxation of supercoiled DNA, a thermodynamically favorable process.
- Work by creating transient single strand break in DNA which allows the DNA on either side of the nick to rotate freely relative to each other.
- uses the other phosphodiester bond as a swivel point
- Resealing is rapid and doesn’t require any addition energy since energy is stored in the phosphotyrosine linkage.
What function does type II topoisomerase have?
- Type II enzymes make a transient double-stranded break in the DNA.
- Activated at sites on chromosome where two double-stranded helices cross each other:
- It uses ATP to:
- 1) break one double-stranded helix reversibly to create “gate”
- 2) causes second strand to pass through
- 3) reseals break and dissociates
- It can separate “decatenate” 2 interlocked DNA circles
- Can prevent severe tangling problems that would arise during DNA replication.
What are the 3 things that type II topoisomerase uses ATP to do?
- Break one double-stranded helix reversibly to create “gate”.
- Causes second strand to pass through.
- Reseals break and dissociates.
What nucleotides make up the nucleotide rich sequence where the replication origin is found?
A-T rich regions where sequence attracts initiator proteins to pry open DNA.
True or False:
Initiation is the only point of control for E. coli, so it’s highly regulated.
True
*proceeds only when sufficient nutrients are present
*refractory perior - delay until new strand is methylated
Why is it enough for bacteri to have only 2 replication forks, but not eukaryotes?
- These two replication forks are enough for bacterial genomes, which are small.
- only 40 minutes to replicate entire genome
- Eukaryotes need more
- traveling at 50 nt/sec, it would take 800 hours to do an average chromosome with a single ORI.
What is the general mechanism of initiation of DNA replication in bacteria?
- Initiator proteins bind to specific sites in ORI, forming complex.
- This complex attracts DNA helicase + helicase loader
- Helicase is placed around a ssDNA exposed by assembly of the complex.
- Helicase loader remains engaged until helicase is properly loaded.
- Helicase unwinds DNA so primase can make RNA primer on leading strand.
- remaining proteins assemble to create 2 replication forks with complexes moving in opposite direction with respect to the ORI.
What phase of the cell cycle does DNA synthesis occur? Also, about how long does it lasts in mammalian cells?
- Occurs during DNA synthesis phase (S).
- About 8 hours for mammalian cells.
True or False:
Chromosomes are replicated to produce two complete copies, joined at centromeres until M phase in the cell cycle.
True
You know that replication is activated in clusters (replication units). But how many replication origins are located in each cluster?
consists of 20-80 replication origins
Is heterochromatin late- or early replicating during S phase?
Late-replicating
- Example: X chromosomes of females:
- almost all of inactive X is condensed into heterochromatin and is replicated late in S phase.
- the active homolog is less condensed and replicates throughout S phase.
- almost all of inactive X is condensed into heterochromatin and is replicated late in S phase.
What are the three minimum requirements for a sequence to be an ORI in yeast?
- must have binding site for ORC (origin recognition complex).
- must have an A-T rich stretch for easy unwinding.
- must have binding site for proteins that help attract ORC.
What proteins bind to form the pre-replication complex and regulate origin activity?
- Helicase
- Helicase loading proteins:
- Cdc6
- Cdt1
In the S phase of the cell cycle, what do activated Cdk proteins lead to?
- dissociation of helicase loading proteins
- activation of helicase
- unwinding of DNA
- loading of DNA polymerase, etc.
What control mechanism prevents assembly of a new ORC until the next M phase?
Once the pre-replication complex is formed, the ORC is phosphorylated to prevent it from replicating a second round before the cell divides. Once the cell divides, then the ORC will be de-phosphorylated to activate it again.
- single chance to form in G1 when Cdk activity is low.
- second window for pre-replicative complexes to be activated and disassembled in S phase when Cdk’s activity is high.
True or False:
ORI function depends critically on distant sequences.
True - if there is DNA deletions in either the ORI or the thousands of nucleotide pair distant sequences, then the indiated origin of replication is inactive.
- Specific human sequences have been identified that can serve as ORIs; they are 1000s of nucleotide pairs in length.
- They can still function if moved to a different locus if placed where chromatin is uncondensed.
- Example: distant DNA in beta-globin cluster is required for expression of the genes in the cluster.
- ORI function depends critically on distant sequences.
- also affects transcription
- global effect of decondensing chromatin structure.
- ORI function depends critically on distant sequences.
For efficient replication, what is needed to destabilize the DNA-histone interface?
Chromatin remodeling proteins.
As the replication fork passes through chromatin, what does the histone octamer break into? Also, which dimers are found on the daughter nucleosomes?
- Breaks into:
- an H3-H4 tetramer, distributed randomly to daughter duplexes
- 2 H2A-H2B dimers which are released from the DNA
Why are histone chaperone proteins needed during reassembly?
- Freshly made H3-H4 fills in spaces; H2A/H2B dimers are half old and half new; they are added at random to complete complex.
- Directed to DNA with sliding clamp called PCNA.
What are the two histone chaperone proteins that we covered in class?
- NAP-1
- loads H2A-H3B dimer
- CAF-1
- loads newly synthesized H3-H4 tetramer
True or False:
Some daughter nucleosomes contain only parental histones or only new ones, but most are hybrids of old and new.
True
Patterns of histone modification can be inherited. Howare these histone modifications spread?
By the reader-writer complexes.
*may be responsible for some types of epigenetic inheritance*
What is the special sequence that is repeated about 1000 times at the end of each chromosome?
GGGTTA
Why is there a end replication problem on the lagging strand? How is this overcame?
- Because there is not a place for an RNA primer.
- Thus, an enzyme called telomerase replenishes these sequences by elongating parental strand in 5’-to-3’ direction using an RNA template on the enzyme.
*Bacteria have circular genomes so they don’t have telomers like eukaryotes have*
What happens after the parental strand has been extended by telomerase?
Replication of the lagging strand can be completed by DNA polymerase, using the extension as a template.
- this mechanism (plus a 5’ nuclease) ensures that the 3’ end is longer, leaving a protruding SS end that loops back and tucks into the repeat (called T-loop).
Why are T-loops important at the end of chromosomes?
- Structures protect ends and distinguishes them from broken ones that need to be repaired.
- Shelterin (protein) - protective chromosome cape made up of proteins.
Do stem cells retain full telomerase activity?
Yes
True or False:
Human somatic cells are born with full complement of telomere repeats.
True
Why are telomere repeats lost after each generation?
Due to insufficient telomerase activity.
What is replicative senescence?
After many generations, daughter cells will have defective chromosomes and stop dividing; in this way the cell’s lifetime is regulated to guard against cancer.
How many times do human fibroblasts normally divide before undergoing replicative senescence?
60 times.
**continue dividing when telomerase is provided experimentally**
What disease in humans is characteristic of the patient carrying a mutant telomerase RNA gene?
- Dyskeratosis Congenita
- develop prematurely shortened telomeres
- die of progressive bone marrow failure
