Molecular Biology Exam 1, DNA are Genes PT 2

3 requirements of DNA replication

1. sequence of nucleotides may encode information about protein assembly (genetic code)

2. changing a base in DNA (mutation) could change the way (code) in which protein is synthesized

3. possible method of replication

what mechanism of DNA replication did Watson and Crick elude to?

semiconservative replication; where each strand of the double helix would serve as a template for synthesis of a new strand

Semiconservative replication

• unzips at one end (unwinding of strands)

• exposed bases on each of the two strands

• exposed bases can potentially pair

  • strict pairing requirements (a-t and g-c)

• the two strands act as templates

• direct assembly of complementary nucleotides to reform a double helix structure, identical to the original

  • each new strand has one parental strand and one newly synthesized strand

what was another prediction of DNA replication based on the Watson and Crick model (aside from semiconservative replication)

replication fork; location at DNA where the double helix is unwound to produce two single strands (which will function as templates for synthesis of a new strand)

Arthur Kornberg

• isolated the first DNA synthesizing enzyme from E coli, DNA polymerase

• DNA pol - add nucleotides to a growing DNA strand, using an existing strand as a template

• awarded Nobel Prize in 1959

why was the discovery of DNA pol important

it provided the first direct evidence that DNA replication is an enzyme mediated process

DNA pol

• enzyme that adds deoxyribonucleotides to the 3-end (OH group) of a growing nucleotide chain

• substrates for this enzyme: dATP, dCTP, dGTP, dTTP

What is the main function of Pol I

removing RNA primers and replacing them with DNA

the 3 enzymatic activities of Pol I

1. 5’-3’ pol activity

2. 3’-5’ exonuclease activity

3. 5’-3’ exonuclease activity

5’-3’ pol activity

• adds nucleotides to the growing DNA strand

• DNA synthesis always occurs 5’→ 3’

3’-5’ exonuclease activity

• removes incorrectly paired nucleotides from the 3’ end of the growing strand

• provides proof reading ability

5’-3’ exonuclease activity

• removes nucleotides ahead of the polymerase including RNA primers during DNA replication

• allows pol I to remove and replace RNA primers with DNA

DNA polymerase III (pol III)

primer DNA-synthesizing enzyme at the bacterial replication fork (ahead of the replication machinery)

DnaB

the enzyme responsible for unwinding DNA double helix

helicase

breaks down hydrogen bonds between complementary bases, separating the two DNA strands

on what end of DNA does DNA pol add new nucleotides

3’ end ; DNA is always synthesized in the 5’→3’

leading strand

synthesized continuously in the same general direction as DNA unwinding; a smooth continuous process; initiated by a RNA primer

lagging strand

synthesized discontinuously in short segments because DNA polymerase can only synthesize 5’→3’ ; is not synthesized continuously and is done in short segments (okazaki fragments); DNA pol III will extend for some time then move back to the replication fork; initiated by a RNA primer for each Okazaki fragment

primer

short nucleotide chain that binds to template (acts as a start site for DNA polymerase; will be complementary to the DNA template and prodvie the 3’ end needed by DNA pol to begin synthesis

primase (DNaG)

the enzyme that makes short RNA primers (about 8-12 nucleotides long)

DNA ligase

seals the remaining gap between DNA fragments by forming a phosphodiester bond; important for joining okazaki fragments on the lagging strand

DNA replication

1. primase synthesis short RNA oligonucleotides (primer) copied from DNA

2. DNA polymerase III elongates RNA primers with new DNA

3. DNA polymerase I removes RNA at 5’ end of neighboring fragments and fil the gap

4. DNA ligase connects adjacent fragments

Steps of DNA Ligase sealing the DNA backbone

1. After DNA pol I removes RNA primers and replaces them with DNA, small breaks remain in the sugar-phosphate backbone

2. DNA ligase seals these breaks by joining adjacent DNA fragments

3. on the lagging strand, ligase joins the 3’ end of one DNA fragment to the 5’ end of the next

4. creates a continuous phosphodiester backbone

under typical conditions, how often to errors occur to DNA

only 1 error occurs for every 10 billion nucleotides added

what is a source of the extremely high fidelity of DNA replication

3’→ 5’ exonuclease activity of DNA polymerase; enzyme has a proofreading function; incorrectly paired nucleotides are detected and removed before DNA synthesis continues

why is it important that DNA has high fidelity

ensures that genetic info is accurately passed from one generation of cells to the next

Replisome

• large nucleoprotein (protein-DNA complex) that carries out and coordinates DNA replication at the replication fork

• brings together many enzymes / proteins needed for rapid, accurate DNA synthesis

• coordinates replication activities so that the leading and lagging strand can be copied simultaneously

Major components of the replisome

DNA helicase, primase, DNA pol III, sliding (beta) clamp, single stranded binding proteins

sliding (beta) clamp

keeps DNA pol attached to DNA

single stranded binding proteins

stabilizes the unwound DNA; prevents DNA from recombining / interacting temporarily; easily dislodged but allow DNA pol to come in and synth. new DNA strands

DNA polymerase III holoenzyme

• the main DNA-replication machine E coli

• large protein complex containing: two DNA pol III catalytic cores

• each catalytic core synthesizes two new DNA strands

  • one core → leading-strand synthesis

  • one core → lagging strand synthesis

• allows both strands to be synthesized simultaneously at the replication fork

beta clamp

• forms a ring around the DNA and acts like a sliding clamp that keeps DNA poly III attached to the template

• without the beta clamp: DNA pol III would only add ~10 nucleotides before dissociating

• with the beta clamp: DNA pol III can add tens of thousands of nucleotides without falling off (processivity)

processivity

the ability to remain attached while synthesizing DNA

beta clamp loader

• the beta clamp must be loaded onto the DNA

• the clamp loader is ATP dependent protein complex that opens and positions the beta clamp around DNA

steps of how the beta clamp is loaded

1. clamp loader binds ATP and interacts with the beta clamp

2. beta clamp opens and the complex recognizes the primer-template junction

3. the clamp loader position the open clamp around the DNA

4. ATP hydrolysis causes the clamp to close around the DNA

5. the clamp loader releases, leaving the beta clamp attached to the DNA

6. DNA pol III binds the beta clamp and begins / continues DNA synthesis

does primase need a clamp like DNA pol III?

no; primase does not remain attached to DNA for long periods, it synthesizes only a short RNA primer (8-12 nucleotides) and the dissociates, this makes primase a distributive enzyme

distributive enzyme

performs a short task then releases from the molecule

what two major enzymes help to manage the opening and closing of DNA’s double helix

helicase and topoisomerase

helicase

• unwinds DNA double helix by separating the two strands (breaks H bonds)

• breaks the hydrogen bonds between complementary bases

• at the replication fork, helicase expresses the single stranded templates for DNA synthesis

topoisomerase

• prevents the excessive twisting and overwinding of DNA ahead of the replication fork

• temporarily cut the DNA backbone, allow the DNA to rotate or pass through itself, and then reseal it

• breaks covalent linkage

eukaryotic DNA replication

• uses the same basic semi-conservative mechanism as in bacteria, with leading and lagging strands

• is considered more complex in eukaryotes than in prokaryotes

  • requires more proteins and regulatory factors at the replisome

why are eukaryotic genomes much larger and more complex than bacterial genomes

• multiple chromosomes

• much greater amounts of DNA

• DNA is packaged into chromatin

How are larger genomes, such as in eukaryotes, replicated in a reasonable amount of time

• replication occurs at many origins simultaneously

• multiple replication bubbles form along each chromosome

How is DNA replication in eukaryotes coordinated with chromatin organization

• nucleosomes must be temporarily displaced during replication

• chromatin must be reassembled behind the replication fork

Eukaryotic origins of replication

• approx. 400 origins disperse throughout the 16 chromosomes of yeast

• estimated to be thousand of origins among chromosomes in the nucleus of the human cell

• in eukaryotes, replication proceeds in both directions from multiple points of origin from each chromosome

telomeres

• repetitive DNA sequences located at the ends of eukaryotic chromosomes

• protects chromosome ends from being recognized as DNA breaks and to help stabilize chromosome structure

• telomeres form a protective looped structure at chromosome ends

• implicate aging as they become shorter with each round of replication; act as a buffer to prevent loss of necessary info from being lost

the end-replication problem

• DNA polymerase cannot completely replicate the 5’ end of the lagging strand

• removal of the final RNA primer leaves a short region of unreplicated DNA

• as a result, telomeres become progressively shorter with each round of replication

shortening of telomeres

• progressive shortening can have detrimental effects on cells

• critically short telomeres can lead to chromosome instability

• can contribute to DNA damage responses, cellular senesence (no longer able to replicate), or cell death

telomerase

• enzyme that maintains and extends telomeres

• adds repetitive DNA sequences to chromosome ends

• helps maintain the protective structure of telomeres