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Advanced Cell Biology. Lecture 13
Advanced Cell Biology. Lecture 13
Alexey Shipunov
Minot State University
February 13, 2012
Advanced Cell Biology. Lecture 13
Outline
Questions and answers
DNADNA replicationDNA reparation
Advanced Cell Biology. Lecture 13
Outline
Questions and answers
DNADNA replicationDNA reparation
Advanced Cell Biology. Lecture 13
Questions and answers
Previous final question: the answer
Why cells use RNA as DNA replication primers?
I Primers should be used for starting nucleotide chain,therefore they should be nucleic acids
I Primers contain errors and should be removed, thereforethey should be distinguishable from the rest of chain,therefore, they should be RNA instead of DNA
Advanced Cell Biology. Lecture 13
Questions and answers
Previous final question: the answer
Why cells use RNA as DNA replication primers?
I Primers should be used for starting nucleotide chain,therefore they should be nucleic acids
I Primers contain errors and should be removed, thereforethey should be distinguishable from the rest of chain,therefore, they should be RNA instead of DNA
Advanced Cell Biology. Lecture 13
Questions and answers
Meselson-Stahl experiment (1958), again
How to rule out two other hypotheses?
Advanced Cell Biology. Lecture 13
DNA
DNA replication
DNADNA replication
Advanced Cell Biology. Lecture 13
DNA
DNA replication
DNA helicases
I DNA helicases are natural zippersI They use energy of ATP to untangle the double helixI Single-strand binding protein (SSBP) associates with DNA
strand to prevent re-forming base pairs
Advanced Cell Biology. Lecture 13
DNA
DNA replication
Single strand binding protein, SSBP
Advanced Cell Biology. Lecture 13
DNA
DNA replication
Sliding clamp (DNA clamp)
I Keeps DNA polymerase attached to the templateI Form a ring around different DNA polymerasesI Most are trimers of PCNA proteins
Advanced Cell Biology. Lecture 13
DNA
DNA replication
Human DNA clamp protein (trimer of PCNA)
Advanced Cell Biology. Lecture 13
DNA
DNA replication
DNA sliding clamp movie
Advanced Cell Biology. Lecture 13
DNA
DNA replication
DNA replication complex
1. DNA: old helix, leading strand, lagging strand2. DNA polymerases3. DNA helicases4. SSBP5. DNA clamp6. Primase7. DNA ligase
Advanced Cell Biology. Lecture 13
DNA
DNA replication
DNA replication machine (simplified)
Advanced Cell Biology. Lecture 13
DNA
DNA replication
DNA replication machine (more realistic)
Advanced Cell Biology. Lecture 13
DNA
DNA replication
DNA replication sewing machine movie
Advanced Cell Biology. Lecture 13
DNA
DNA replication
DNA replication in general movie
Advanced Cell Biology. Lecture 13
DNA
DNA replication
Telomerase
I Lagging strand cannot reach the end of DNA moleculeI Every replication cycle chromosome lost parts of
telomeres from its endsI To prevent a loss on meaningful DNA fragments,
telomerase extends chromosome with new telomeresequences
I In humans, telomeres are several thousands of TTAGGGsequences
I Telomeres are also recognizable ends of chromosomes
Advanced Cell Biology. Lecture 13
DNA
DNA replication
Telomerase and telomere
Advanced Cell Biology. Lecture 13
DNA
DNA replication
Telomere movie
Advanced Cell Biology. Lecture 13
DNA
DNA replication
Telomere theory of aging
I In humans, telomerase is only active in germ cells, stemcells and certain white blood cells
I Olovnikov (1971) postulated that lost of DNA ends willeventually stop division of cells and may stimulatesenescence of cells
I There is a strong support of this hypothesis with some celltypes (e.g., blood vessels wall cells); however, mice withknocked-out telomerase gene do not show significantlyless lifespan
Advanced Cell Biology. Lecture 13
DNA
DNA reparation
DNADNA reparation
Advanced Cell Biology. Lecture 13
DNA
DNA reparation
Single nucleotide mutations
I SN mutations (deletions and substitutions) may damageseverely the function of protein
I In sickle-cell anemia, substitution turns Glu to Val andmodify structure of hemoglobin and then red blood cells
I However, people with partly modified red blood cells areless susceptible to malaria
Advanced Cell Biology. Lecture 13
DNA
DNA reparation
Sickle-cell anemia
Advanced Cell Biology. Lecture 13
DNA
DNA reparation
Mutation theory of ageing and/or cancer
I Accumulation of mutations will result in a constant loss offunctions
I Cells will either degrade or start to go out of control(cancer)
Advanced Cell Biology. Lecture 13
DNA
DNA reparation
DNA mismatch repair
I Normally, error rate of DNA polymerase + proofreading is≈10−7
I DNA mismatch repair proteins decrease it to ≈10−9
I They react on DNA conformation deviations; recognizenewly synthesized strand by nicks, and remove wrongfragments which are later replaced with DNA polymeraseand ligase
I Some cancers are results of mutations in DNA mismatchprotein genes
Advanced Cell Biology. Lecture 13
DNA
DNA reparation
DNA mismatched repair system
Advanced Cell Biology. Lecture 13
DNA
DNA reparation
Depurination and deamination
I Depurination is detaching A and G from its sugar; it resultsin deletion*
I Deamination is C to U conversion; it results in substitution*
Advanced Cell Biology. Lecture 13
DNA
DNA reparation
Depurination and deamination
Advanced Cell Biology. Lecture 13
DNA
DNA reparation
Final question (1 point)
What is the difference between deletion and substitution?
Advanced Cell Biology. Lecture 13
DNA
DNA reparation
Final question (1 point)
What is the difference between deletion and substitution?
Advanced Cell Biology. Lecture 13
DNA
DNA reparation
Summary
I DNA replication system is a multienzyme complexI Ends of chromosomes are constantly shortening and
extending with new telomeresI DNA suffers from multiple damaging events; multiple
reparation systems are trying to lower mutation risks
Advanced Cell Biology. Lecture 13
DNA
DNA reparation
For Further Reading
A. Shipunov.Advanced Cell Biology [Electronic resource].2011—onwards.Mode of access: http://ashipunov.info/shipunov/school/biol_250
B. Alberts et al.Essential Cell Biology. 3rd edition.Garland Science, 2009.Chapter 6.