Advanced Cell Biology. Lecture...

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

DNA helicase movie

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.