Advanced Cell Biology. Lecture 12 -...

Advanced Cell Biology. Lecture 12

Advanced Cell Biology. Lecture 12

Alexey Shipunov

Minot State University

February 10, 2012

Advanced Cell Biology. Lecture 12

Outline

Questions and answers

ProteinsHow proteins are studied

DNADNA replication

Advanced Cell Biology. Lecture 12

Outline

Questions and answers

ProteinsHow proteins are studied

DNADNA replication

Advanced Cell Biology. Lecture 12

Outline

Questions and answers

ProteinsHow proteins are studied

DNADNA replication

Advanced Cell Biology. Lecture 12

Questions and answers

Previous final question: the answer

Which way of sedimentation employs differences betweenmolecular sizes?

I Velocity sedimentation: larger proteins sediment faster

Advanced Cell Biology. Lecture 12

Questions and answers

Previous final question: the answer

Which way of sedimentation employs differences betweenmolecular sizes?

I Velocity sedimentation: larger proteins sediment faster

Advanced Cell Biology. Lecture 12

Questions and answers

Teams

I Studies on the chemical nature of the substance inducingtransformation of Pneumococcal types. — Avery, O.T.,MacLeod, C.M. & McCarty, M.J. — Exp. Med. 79, 137-159(1944)

I Molecular Structure of Deoxypentose Nucleic Acids —Wilkins M.H.F., A.R. Stokes A.R. & Wilson, H.R. —Nature171, 738-740 (1953)

I (1) A Structure for Deoxyribose Nucleic Acid — WatsonJ.D. and Crick F.H.C. — Nature 171, 737-738 (1953) AND(2) Genetical Implications of the structure ofDeoxyribonucleic Acid — Watson J.D. and Crick F.H.C. —Nature 171, 964-967 (1953)

Advanced Cell Biology. Lecture 12

Proteins

How proteins are studied

ProteinsHow proteins are studied

Advanced Cell Biology. Lecture 12

Proteins

How proteins are studied

X-rays crystallography

I Based on the ability of proteins to form crystalsI These crystals will scatter X-rays and we will see difraction

pattern, different for different proteinsI Useful for prediction of protein 3D structure

Advanced Cell Biology. Lecture 12

Proteins

How proteins are studied

X-rays crystallography

Advanced Cell Biology. Lecture 12

Proteins

How proteins are studied

Nuclear magnetic resonance (NMR)

I Unfortunately, not all proteins form crystals and it is notalways possible to obtain enough protein for crystallization

I Method is based on the response of nuclei to radio wavesI Again, it is used mostly for understanding conformation of

protein

Advanced Cell Biology. Lecture 12

Proteins

How proteins are studied

Nuclear magnetic resonance

Advanced Cell Biology. Lecture 12

Proteins

How proteins are studied

Protein databases

I UniProtI NCBI proteinI RCSB

Advanced Cell Biology. Lecture 12

Proteins

How proteins are studied

NCBI Protein database

Advanced Cell Biology. Lecture 12

Proteins

How proteins are studied

RCSB database

Advanced Cell Biology. Lecture 12

Proteins

How proteins are studied

UniProt database

Advanced Cell Biology. Lecture 12

DNA

DNA replication

DNADNA replication

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Template

I Both DNA strands may act as a template for the synthesisof other strand

I Template hypothesis was first expressed by Nikolaj Koltsovin 1927

I Replication is semiconservative

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Meselson-Stahl experiment (1958)

I Theoretically, three variants of replications were possible:semiconservative, dispersive and conservative

I Experiment was based on two bacterial cultures whichgrew on different media: with normal nitrogen 14N, andwith heavy nitrogen, 15N

I After growing for 20’ on heavy medium, bacteria produceDNA molecules with intermediate weight only

I That ruled out conservative hypothesis.

How to rule outdispersive hypothesis?—Explanation has been given onlecture.

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Meselson-Stahl experiment (1958)

I Theoretically, three variants of replications were possible:semiconservative, dispersive and conservative

I Experiment was based on two bacterial cultures whichgrew on different media: with normal nitrogen 14N, andwith heavy nitrogen, 15N

I After growing for 20’ on heavy medium, bacteria produceDNA molecules with intermediate weight only

I That ruled out conservative hypothesis. How to rule outdispersive hypothesis?—Explanation has been given onlecture.

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Replication origin and forks

I DNA double helix should open for replication: this is areplication origin place

I When replication starts, these openings will grow and formreplication forks which are visible under microscope

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Lagging and leading strands

I DNA is synthesized only in 5’-to-3’ direction. Structureof DNA and complexity of replication do not allow the otherdirection.

I Therefore, fork is asymmetrical: one strand is replicatingsmoothly whereas other strand (lagging) replicated by“leaps”, still 5’-to-3’

I Every “leap” produce one Okazaki fragment which are laterjoined

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Advanced Cell Biology. Lecture 12

DNA

DNA replication

DNA proofreading

I Every time DNA polymerase adds new nucleotide, itchecks if previous was correctly placed (if not, it removesthe wrong nucleotide)

I Two different binding sites in DNA polymerase work forsynthesis and proofreading

I Proofreading goes in opposite, 3’-to-5’ directionI As a result, DNA polymerase enzyme error rate became

less than ≈0.00000001

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Advanced Cell Biology. Lecture 12

DNA

DNA replication

DNA polymerase movie

Advanced Cell Biology. Lecture 12

DNA

DNA replication

RNA primers

I DNA polymerase cannot start nucleotide chain itselfI Instead, primase enzyme synthesize small RNA primer

(≈10 nucleotides) which used as a starting pointI Primase error rate is ≈0.0001 (!)I In lagging strand, RNA sites are interleaving with DNA

fragmentsI Then RNA sites are erasing, and DNA ligase joins

fragments together

I Why primer is RNA?

Advanced Cell Biology. Lecture 12

DNA

DNA replication

RNA primers

I DNA polymerase cannot start nucleotide chain itselfI Instead, primase enzyme synthesize small RNA primer

(≈10 nucleotides) which used as a starting pointI Primase error rate is ≈0.0001 (!)I In lagging strand, RNA sites are interleaving with DNA

fragmentsI Then RNA sites are erasing, and DNA ligase joins

fragments togetherI Why primer is RNA?

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Final question (3 points)

Why cells use RNA as DNA replication primers?

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Final question (3 points)

Why cells use RNA as DNA replication primers?

Advanced Cell Biology. Lecture 12

DNA

DNA replication

Summary

I DNA replication is a semiconservative processI DNA replication could go only in one directionI Proofreading and RNA priming are helping in replication

Advanced Cell Biology. Lecture 12

DNA

DNA replication

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, pages 198–208.