March 10 th and 12 th , 2010 Biochemistry Recitation MBioS 303 - - PDF document

3/12/2010 1 MBioS 303 Spring 2010

March 10th and 12th, 2010 Biochemistry Recitation Objectives

1)

To understand what it means to say that DNA replication is semi-conservative

2)

To understand the process of DNA replication, including all involved proteins

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Objectives

1)

To understand what it means to say that DNA replication is semi-conservative

2)

To understand the process of DNA replication, including all involved proteins

Replication Conservative or Semi-Conservative??

Genomes, 2nd Ed.

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Objectives

1)

To understand what it means to say that DNA replication is semi-conservative

2)

To understand the process of DNA replication, including all involved proteins

DNA Replication Bidirectionality

1)

Replication is bi-directional (proceeds in two directions at once)

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DNA Replication General Principles

1)

Replication is bi-directional (proceeds in two directions at once)

2)

New strands are always synthesized in the 5’ 3’ direction (meaning template strands are read in the 3’ 5’ direction)

DNA Replication Addition of dNTPs

Molecular Biology of the Cell, 4th Ed.

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DNA Replication Addition of dNTPs

How does DNA Polymerase know what base to add next?

DNA Replication Addition of dNTPs

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DNA Replication General Principles

1)

Replication is bi-directional (proceeds in two directions at once)

2)

New strands are always synthesized in the 5’ 3’ direction (meaning template strands are read in the 3’ 5’ direction)

3)

One strand is synthesized continuously, the other is synthesized discontinuously

DNA Replication Continuous vs. Discontinuous

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DNA Replication Required elements

1)

Template – why???

2)

Primer (short oligonucleotide that is complementary and hydrogen bonded to template strand) with free 3’ OH group – why???

3)

Lots of enzymes (over 20 in E. coli)

DNA Replication A Closer Look

• DNA Replication consists of three stages:

1.

Initiation

2.

Elongation

3.

Termination

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DNA Replication Initiation

• Bacteria generally have only one circular

chromosome

• In E. coli, there is one origin of replication on this

chromosome

Contains five binding sites for DnaA ATPase Containes three adjacent AT rich sequences (DUE)

DNA Replication DnaA

• Binding of DnaA results in supercoiling
• Supercoiling compensated for by relaxation in DUE
• Results in single-stranded template

Genomes, 2nd Ed.

What binds the single-stranded template, and why?

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DNA Replication DnaC

• ATPase
• Loads DnaB

DNA Replication DnaB

• Helicase
• Moves along single-stranded template in 5’ 3’ direction, creating

supercoiling in front (relieved by topoisomerase II, aka DNA gyrase)

• One DnaB complex at each replication form
• Linked directly or indirectly to all other proteins in replication fork

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DNA Replication Elongation

• Leading strand synthesis and lagging strand

synthesis differ

• What’s the same between the two?

1.

DNA helicases unwind parent DNA

2.

Topoisomerases relieve topological stress

3.

Each separated strand stabilized by single-stranded DNA binding proteins

4.

Chains elongated by DNA polymerase

DNA Replication DNA Polymerase

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DNA Replication Leading Strand Synthesis

Molecular Biology of the Cell

1.

Short RNA primer synthesized by primase

2.

DNA polymerase core subunits (linked to DnaB) add dNTPs to growing strand in 5’3’ direction

3.

Strand synthesis proceeds continuously

DNA Replication Lagging Strand Synthesis

1.

A single DNA polymerase complex is used at each replication fork, complicating this process

• One of DNA core subunits used for continuous

leading strand synthesis

• Second core subunit cycles from one Okazaki

fragment to the next

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DNA Replication Lagging Strand Synthesis DNA Replication

To see the movie that was here (along with a couple of other good videos) check out the ‘Useful Links’ tab on the webpage.

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DNA Replication Prokaryotes vs. Eukaryotes

• Prokaryotes: one origin of replication
• Eukaryotes: multiple origins of replication (why?)
• Eukaryotes have linear chromosomes, not circular.
• What problem does this cause? Why?
• How do the cells solve it?

Have a great spring break!